TW321685B - Gene expression system comprising the promoter region of alpha-amylase gene - Google Patents

Abstract

It has been found that during the suspension culture of rice cells, when the culture medium was depleted of sugar (sugar starvation), an excessive gene expression of some alpha-amylase genes located in the cultured cells takes place, resulting in the mass production of alpha-amylase, which is subsequently secreted out of the cell and into the culture medium. In the present invention, DNA fragments which comprise the alpha-amylase gene promoter and DNA sequence encoding the signal peptide chain of the enzyme, and which is capable of being induced to conduct extensive gene expression under sugar depletion, were screened from rice genomic DNA library. The alpha-amylase gene promoter and the DNA sequence encoding the signal peptide chain together with glucuronidase reporter gene and hygromycin resistance gene were then utilized in the construction of a GUS gene expression vector, which was in turn transformed into the rice suspension-cultured cells, so as to investigate the expression of the GUS expression vector in the cells under the control of the alpha-amylase promoter. In such a way, it is possible to conduct gene regulation and protein expression and secretion by utilizing the characteristics of the alpha-amylase gene promoter and the DNA sequence encoding the signal peptide chain.

Description

321685 Α7 Β7 Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of invention (/) Starch contains two glucose polymers, amylose and amylopectin, and is the most important nutrient storage component of cereal seeds (AKazawa et al., 1985). At the early stage of germination of cereal seeds, the silty cell will synthesize α-starch hydrolase, 〇t-starch hydrolase-glycosidase and limit the secretion of dextrinase and other enzymes into the endosperm to hydrolyze starch together to form glucose and maltose to provide embryos Nutrients required for development (Rogers, 1985). The enzyme involved in the hydrolysis of starch still has a / 3-starch hydrolase, which can hydrolyze starch into maltose and a small part of glucose. Usually, / 3-starch hydrolase binds to the proteosome in the form of an inactive form in the endosperm in the dried seeds (Tronier et al., 1970). When the seeds germinate, the aleurone cells are also induced by GA3 to produce proteases, which can break the disulfide bond and release the active form; 3-starch hydrolase. i The above four enzymes are involved in starch hydrolysis during seed germination, but the most produced by α-starch hydrolase and plays the most important role (Akazawa et al., 1988) * Among them, GAa is known to ot +- The performance of the source powder hydrolase has a direct shadow of honey (Chandler et al., 1987). When rice seeds are treated with GA3, the newly synthesized α-starch hydrolase mRNA will be increased by 50 to 100 compared with the normal situation. Times (O'Neill et al., 1990). In fact, the large amount of expression of the α-starch hydrolase mRHA induced by GA3 has provided an ideal research model for how plant hormones regulate gene expression (Ho et al., 1987). The ot-starch hydrolase genes of rice, barley and wheat have been selected in large numbers and further research and analysis have been carried out. The results show that these cereals ~ _ class α-starch hydrolase isomerases are composed of a variety of α-starch Please read the notes on the back of the hydrolase gene 14 to fill out this page) -The paper size of the binding and binding book is applicable to the Chinese National Standard Falcon (CNS) Α4 specification (2 丨 0χΜ7mm) 4 321685 A7 Central Standards Bureau staff of the Ministry of Economic Affairs Printed by the consumer cooperative B7 V. Description of the invention (part 1) Manufactured (Baulcombe et al., 1987; Huany et al., 1990; Knox, 1987) 0 Barley and wheat seeds are secreted by aleurone cells during hair wear The resulting α-starch hydrolase is divided into two categories: high isoelectric point and low isoelectric point (Jacobsen, 1982; Lazarus et al., 1985). There are about 7 ct-starch hydrolase genes in barley belonging to high isoelectric points, and 3 to 4 tilt genes belong to low isoelectric points (K ^ ursheed &amp; Rogers, 1988). So far, there are 7 types of ot-starch in barley. Hydrolase cDNA and DNA of nine α-starch hydrolase gene groups have been selected (Chandler et al., 1984; Deikman et al., 1985; Khrusheed &amp; Rogers, 1988; Knox et al., 1987; W hi 11 ie η eta 1., 1 9 8 7). The a-house powder hydrolase gene in wheat is divided into three groups: 〇11 <1, 〇 [-/ \ | 〇72′〇 (-& 111 YA3. 〇 (-/ \ | 1171 has high Electrical point, ct-Amy2 is a low isoelectric point, they each contain at least 10 ® genes and appear in germinated seeds (Colin et al., 1985; Alisonet al., 1988), ct-Amy3 contains 3 Up to 4 genes and only appear in immature seeds (Baulcombe et al., 1987). Research on the rice ct-starch hydrolase gene failed to divide the α-starch hydrolase gene like barley and wheat It is a high isoelectric point group and a low isoelectric point group. In fact, Macgregor et al. (1988) used isoelectric point electrophoresis analysis and found that rice α-starch hydrolase isomerase is concentrated in the PI value of less than 5. 5 position, so rice may not have a high isoelectric isomerase. Huang et al. (1990b) used an interactive hybridization experiment to divide the 10 kinds of rice α-starch hydrolase genes into 5 groups and determined their distribution in 5 Chromosome—on (Ranjhan et ai., The manuscript is in preparation O'Neill et al. (1990) This paper scale applies the Chinese National Standard (CMS)) A4 size (210X297mm) (please read the notes on the back ^ 4 fill in this page first). Pack. Order 321685 printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. A7 ___B7__ V. Description of Invention (彡) First of all, rice α- Starch hydrolase cDNAs P0S103 and P0S137 have been studied in more detail. The molecular weight of the alpha-starch hydrolase precursor protein produced by P0S103 and P0S137 is about 48KDa. When it is secreted out of the cell, the signal of this precursor protein is peptide The bond is cut off, so the molecular weight of the mature starch hydrolase is about 45 to 46 KDa and its isoelectric point is predicted to be about 6.0. However, Kumagai et al. (1990) transferred POS103 into yeast cells. The α-starch hydrolase is secreted into the medium, and it is found that the molecular weight of ex-starch hydrolase is about 44 to 45KDa, and the isoelectric point is about 4.7 to 5.0. The secretion mechanism of the plant protein is broader than that, and there is no deep understanding so far. It may be similar to the secretion mechanism of animals. Secreted proteins are first synthesized by ribosomes on the rough, endoplasmic reticulum (RER) membrane, and the function of the peptide chain i will extend the protein in the signal. Guide into the inner cavity of the endoplasmic reticulum (ER). After the signal peptide is excised, the ER forms a vesicle in a piercing way to contain the protein, and then transfer to the Golgi body to bind it and send the protein. Into the body of Golgi. In the Golgi, it is transferred to the cell membrane by secretory vesicles, and the protein is secreted out of the cell after fusion of the membrane (Walter et al., 1984). Many secreted proteins have been found to be N-glycosylated. It remains unclear what role glycosylation plays in protein secretion. It is known that neither the secreted α-releasing hydrolase in the aleurone cells of barley and wheat is glycosylated (Jacobsen et al., 1988; Tkachuk &amp; Kruger, 1974). Akazawa et al. (1985) have proved that the α-starch hydrolase produced in the blastoderm (scu te 1 1 um) during the perforation period is all secreted proteins that are sugar-based. However, 0 · He ii 1 and others (1990) believe that the Chinese national standard (CNS) A4 specification (210X297mm) is applicable to the paper scale of this paper 6 — ^ ------ ^-(please read the notes on the back first Things to write this page), va 321685 A7 B7 Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. Description of Invention (Mo) There are at least two mature α-starch hydrolases produced during the wearing of rice seeds It is not glycosylated, respectively P0S137 and λ OSgl 'because there is no glycosylation position on the amino acid sequence. Akazawa et al. (1986) found that during the formation of α-starch hydrolase, after the signal peptide chain was removed, an oligosaccharide with a molecular weight of 290 Da was bound to cc-starch hydrolase. Treatment of blastoderm cells with tunicamycin, an inhibitor of glycosylation, revealed that it inhibits glycosylation but not the secretion of α-starch hydrolase (Akazawa &amp; Nishimura, 1985). Therefore, the secretion of cereal α-starch hydrolase may not be related to glycosylation. The wheat and rice α-starch hydrolase genes were transferred into yeast cells, and it was found that wheat α-starch lysozyme was not glycosylated and its secretion was not large (Roths te in et al., (1987) However, rice ct-starch hydrolase is not only glycosylated and secretes a large amount of cx-starch hydrolase (Kumasai et al., 1990), so Kumagai et al. Believe that glycosylation has nothing to do with protein secretion. However, it will affect the amount of α-starch hydrolase secreted by cereals. The secretion of α-starch hydrolase in cells of barley aleurone layer is also affected by calcium ions (Bush et a I., 1989). Α-starch Hydrolase is a metalloprotein containing ions. If the ion is lacking, α-starch hydrolase will lose its enzyme activity and become unstable. GA3 can regulate calcium ion into the ER lumen to hydrolyze with a-starch Enzymes are combined, so GA3 may also regulate the level of post-translation of ct-starch hydrolase synthesis (Bush et a 1., 1989). Using gene transfer technology, using single cells as materials, the expression and function of eukaryotic genes 'S analysis is To study the mechanism of gene regulation in higher organisms, please read the notes on the back kC and fill out this page-installation. The paper size of the binding is applicable to the national standard of the ten countries (CNS) A4 specification (210X 297 mm) shirt 1685 A7 B7 Central Standards Bureau Printed by Beigong Consumer Cooperative. V. Description of the invention (_r) The ideal state of differentiation is faster than that of a treatment. Breeding and molecular plant or plant body control mechanisms are faster than the treatment response. This involves the invention of this case. Genes are also secreted at the same time. Increase the gene diameter and mechanism of the sifter and signal powder hydrolase in the storage, and use the tradition of plant fines, yeasts, etc. The symptom is caused by it ... It is the current system. Plant suspension culture cells have a fast growth rate, consistent, easy to separate protoplasts, easy operation and external treatment, so it is to study plant physiology, biochemistry, transfer, and physical aspects. Very ideal material. Relatively, a specific tissue of the whole tree is used as a material to study the complex physiological and biochemical reactions of gene expression, so it is not easy Research. In the past two years of research, it is known that the presence or absence of sucrose in the cell base of rice suspension culture can control some CC-starch water. In the case of lack of sugar in the culture medium, the hydrolysis of α-starch is greatly stimulated, cc- Synthesis and analysis of starch hydrolase. In this case, it is expected that the DN Α sequence of the peptide-opening bond of the α-starch hydrolase gene from the rice genome DNA regulated by sucrose is expected to further study the performance of sugars on α-amylase The regulation mechanism and the way of secretion of ot-starch hydrolase can then use this new mechanism in the transfer engineering technology. The production of genetically engineered proteins by capsular cells has advantages that are lacking using bacterial methods. For example: Cells are higher nucleated cell organisms, and the resulting protein has many complex post-translational modifications to make the protein live in bacteria but lacks post-translation. The system is limited in terms of yeast; Bacuoovirus is a virus of insect cells. The use of recombinant proteins usually has excellent activity. Therefore, the gene expression system generally used in this system, but the virus must be infected Please-first, read the back page and fill out this page. The bound paper is again applicable to the Chinese National Standard (CNS) A4 specification (210X 297mm) 8 A7 B7 Printed by the Beigong Consumer Capital Cooperative, Central Standards Bureau, Ministry of Economic Affairs (彡) Insect cells cultured can only be used for the preparation of genetically-recombinant proteins, and the culture medium for insect cells must contain serum at a very low cost of 0. · 3. Using the newly developed α-starch hydrolase gene expression bar System, the protein produced can suddenly increase its synthesis amount under the condition of lack of sugar in the culture medium, even more than 50 times, and the White matter may be secreted into the culture i can be simplified purification procedures. &quot; In the research of the inventors in the past two years, it has been found that the rice α-starch hydrolase gene family Ye belongs to two different types of regulation: (1) hormone regulation through seeds, and (2) cells in culture In the metabolic inhibition of useful sulfonate nutrients. It is known that the performance of ct-amyl hydrolase Gong Yin in the deembryo rice seeds is pseudo-induced by sibberel 1 ic acid. On the other hand, the performance of the α-starch hydrolase gene in floating-cultured cells is induced by the lack of sulfonate nutrients. The induction of α-starch hydrolase mRNA in a medium lacking sucrose requires a lag period of 2 to 4 hours. The induction of the expression of the ct-starch hydrolase gene is unusually high and the level of the α-starch hydrolase mRNA can increase to 8 to 20 times after 24 hours of lack of sucrose. The synthesis and secretion of ct-starch hydrolase also depend on the level of sulfonate source. The de-inhibition or inhibition of ct-starch hydrolase synthesis can be reversed by stripping sucrose from the medium or supplementing the medium with sucrose, respectively. The synthesis of α-starch hydrolase by glucose and fructose also exerts an inhibitory effect similar to that produced by sucrose. (Su-May Yu et al., &Quot; Metabolic Depression of a -amylase Gene Express ion in Suspens ion-cu 1 tured • C e 1 1 sof R 丨 ce, ”will be published in" L__B-.. L.0 1 , _C hem. V ο l 266. (Please read the precautions on the back first ίν% to write this page • Install-, τ line paper size is applicable to the national standard falcon (CNS) Α4 specifications (2HTX 297 mm) 9 Economy A7 -____ B7_ Printed by the Ministry of Central Standards Bureau Employee Consumer Cooperative V. Description of Invention (Xi) 1991 Nov.; Su-May Yu et al., &Quot; Catabolic Depression of a -amylase Gene Express ion in Suspension-cultured Cel Is of Rice, "Preparation for publication, this article and other contributions are incorporated into this case as a reference). Using gene transfer technology to transfer DNA into plant cells has become the most common and best way to study the regulatory mechanism of genes. Transplanting DNA into plant cells or tissues, in addition to using the Agrobacterium Ti plastid (Bevan &amp; chilton, 1982) method, there are two chemical methods and physical methods. The chemical method includes the use of PEG method (Krens et al., 1982), poly-L ornithine method (Dav_ey et al., 1980) and basal acid 15 method (Hain et al., 19 85). These chemical drugs are all It is toxic to cells, so it may cause cell damage and affect the efficiency of colonization. Physical laws include electrical penetration method (Fromm et al., 1986), obvious injection method (Crossway et al., 1986), particle impact method (Klein et al., 1987) and ultrasonic method (Joersbo & Brunstedt, 1990), etc., the principle of which is to use mechanical force to create holes in the cell surface to facilitate the entry of DNA, so it will also Causes some degree of damage. In addition, the transferred plastid DNA should carry a resistance gene and a reporter gene. The resistance gene can be used as a transgenic plant because its product can resist the action of certain antibiotics or drugs. Selectable markers. There are two types of resistance genes commonly used by plants, one is the Hygromycin B (Hygromycin B) resistance gene (Waldron et al., 1984). It can inhibit the effect of hygroside _ •. The other is the neomycin phosphotransferase E (NPT H) gene (Herrera et please read the notes on the back WC% to write this page. Install.-° The size of the paper is applicable to the national standard Falcon (CNS) A4 specification (210X 297 mm) 321685 A7 B7 5. Description of the invention (f) Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (al., 1983), which can suppress the effects of Jcanaiaycin and G41S, but many Undergraduate plants are inherently highly resistant to kanamycin, so using HPT II as a selectable marker may make it difficult to determine whether the transformation is successful (Hauptmann et al., 1988; Potrykus et al., 1985). Reporter genes are genes that are used as coding regions during gene fusion, and their transcription and translation functions are directly controlled by the gene itself or foreign gene promoters (Jeferson, 1987). There are t types of reporter genes that are more commonly reported in higher plants, namely / S-galactosidase (UCz) (Helmer et al. 1984) and chloramphenicol acetyltransferase (CAT) (Gorman et al_., 1982 ), Neomycin phosphotransferase (HPTH) (ReiSS et al., 1984), blue eosin (nop ^ aline) synthase (MOS) (BeraI et al., 1983), octopine synthase (OCS) ( DeGreve et al., 1982), firefly luciferase (Ow et al., 1 986), and E. coli / 3-¾-glucuronidase (GUS) (.Jefferson et al., 1987). Among the above seven reported genes, the plant itself produces lacz enzymes, and analysis of enzymes such as NOS, 0CS, and firefly luciferase is more troublesome. At present, the more popular CAT and NPT II dentin analysis also has the disadvantages of being expensive and difficult, and the esterase, phosphoesterase and transferase contained in the plant itself will compete with the CAT and NPT 1 substrates, so Relatively reduces the sensitivity of enzyme analysis (Gorman et al., 1982; Reiss et al., 1984) ° The GUS gene has been widely used as a reporter gene, although Hu et al. (1990) found it in the development of seed plants Both the fruit and immature seeds can measure the strong G US activity, but the G US analysis method is simple and quick to analyze. Please read the notes on the back first I- Binding line paper size is applicable to China National Standard Falcon (CNS) A4 Specifications (210 X 297 mm) 11 A7 B7 Printed by the Employees ’Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy V. Description of invention (V» Acceptance of GUS (5-bromo-4-chloro-3-indolyl glucuronic acid Glycoside (X-gluc) is cheaper, GUS has good stability, and most plants have low GUS activity (j ef fers on, 1987) waiting, so the GUS gene is currently used as a report for higher plants Genetically optimal In view of the purpose &gt; ·-刖 ct-starch hydrolase has been widely used in industry, so 9 can be obtained in a large and simple way to produce this enzyme for the industry needs »It became necessary. Therefore, this case The inventors found that by causing the medium to lack sugar to induce the expression of the α-starch hydrolase gene, the above-mentioned giant can be achieved. Further, the gene expression of the α-starch hydrolase is used in a systematic manner under the condition of sugar deficiency Large amounts of other desired proteins integrated into the gene expression system can be obtained. Therefore, one purpose of this case is to provide a method for generating a large amount of α-starch hydrolase. Another purpose of this case is to provide a novel gene expression bar It is used to manufacture proteins for repatriation engineering for mass production of the desired protein. The present invention will be further illustrated with the following examples and accompanying drawings. However, as those skilled in the art understand, the following examples are only for Ye Describe the invention of this case, not to limit the invention. Materials and 77 Μ I. Screening of the ct-starch hydrolase gene from the rice genome DHA repository 1. Introduction to the rice genome DHA repository: 1. The rice genome DNA library used in this case was purchased from CLONTCH (cat »f FL 1040 D, Lot t »7081), the rice variety used is R 9 tiv ^ L • Japonica. Genome DNA is extracted from 7-day-old rice seedlings in the dark and then made into a genome DNA library. The size of this paper is in accordance with Chinese National Standard (CNS) Α4 specification (210X297mm) Please-read first, read the note binding line A7 B7 5. Invention description (/ P) 2. The stocking carrier of this repository is The EMBL-3 * colonization site is Bam HI, and the average DNA size of the selected colony is 15 kb. This DNA fragment can be isolated from phage DNA by the action of Bam HI or Sal I. 3. For the purpose of screening gene bank, the host bacteria of this EMBL-3 bank is Escherichia coli NM538 or LE 392, which can produce large plaques on the broccoli culture medium. 1.2 Method for screening α-starch hydrolase from the DNA bank of rice genome · &quot; 1. 2. 1. Preparation and infection of bacteriophage host bacteria: 1. Add 5ml LB liquid medium (per liter) to a 10ml test tube Contain 10g of NaCl, 10g of pancreatic gland, 5g of yeast extract), pick a single colony of E. coli NM538 with a toothpick into this test tube, shake it at 37 ° C and keep it for about 6 ~ 8 hours until the ODe &quot; value is 0.5 ~ 0.7. 2. Centrifuge at 3000 rpm for 10 minutes using a Sorvall RT6 000B centrifuge at 4 ° C, and dissolve the precipitated bacterial bodies with ice-cold 10 mM MgSO * (add 3 ml of MgS〇4 per 10 ml of bacterial cell precipitate to dissolve ), Store it at 4 ° C for future use (about one week for the Ministry of Economic Affairs, Central Standards Bureau, Beigong Consumer Co., Ltd. Du Printing 3. Dissolve 80ml of Top agar in a wave oven (plus XlM per liter of LB liquid medium) MgS〇4 10ml, 10N NaOH 0.2ml, agar 8g), divide it into 8 tubes, each containing 9m 1 of Top agar, and keep it in a water bath at 46 ° C. 4. Transfer 6m1 of rice gene Group storage (potency 2x104 / jU) and the prepared 2. 4ml NM538 bacterial solution are thoroughly mixed, and cultured in 37t for 15 ~ 20 minutes. '5 · Take 300ra 1 of the mixed bacterial solution in sequence, add this The size of the paper in the 8-tube Top agar applies the Chinese National Standard (CNS) 84 specifications (210 X 297 mm) 321685 A7 ____B7___ printed by the Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economy V. Description of invention (//) Cover tightly Cover the test tube, then shake up and down to mix thoroughly, and quickly pour into a large petri dish (diameter 14cm) pre-warmed at 37 ° C ) On the solid medium (1 liter of LB liquid medium plus 15g agar), wait for Top agar to coagulate, and then put the 48-value petri dish upside down in the 37 υ incubator for overnight cultivation, the next day you will see densely covered plaques. 1.2.2 .Transfer the DNA in the lysate to the nitrocellulose filter paper: • 1. Prepare 3-value shallow plates, each with a Whatman 3M filter paper on top, and three shallow plate filter papers with denaturing solution (0.5M NaOH + 1.5M NaCl), neutralizing solution (4.5M NaCl + l.OM Tris-HCl, pH 8.0) and 2xSSC (each containing 17.5g NaCl and 8.8g sodium acetate) are fully moistened. Wet it. 2. On the base fiber Mark the surface of the (NC) filter paper with a signature pen, and gently cover the solid medium covered with plaque B with the mark face down, and then use a pin to place asymmetrical points on the medium to pick plaque pairs in the future. Suitable for use 3. Slowly tear the NC filter paper from the culture medium, with the mark side facing up, place it on the denaturing solution for 5 minutes, then transfer to the neutralizing solution for 5 minutes, and finally react in 2xSSC for 2 minutes. 4 . Put the NC filter paper on the 3MM filter paper, let it dry, and then bake it in the 80 ° C oven for 2 hours. 1. 2. 3 .Use probe hybridization to select "-starch hydrolysis ... Enzyme gene: 1.2.3.1. Agar colloid recovery DNA (Maniatis et al., 1989):" 1. Rice α-starch hydrolase cDNA pure RMAYC30 (Tzou, 1990) | Binding (please read the notes on the back first and fill in this page) (This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297mm) -14-A7 B7 Ministry of Economic Affairs Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards 5. Description of the invention (/ &gt;) After the restriction enzyme EcoR I is applied, a loading buffer of 0.15 times the volume (0.25¾ bromophenol blue, 0.25% 2%) is added Toluidine (cyanol), 30¾ glycerin). 2. Using agar colloid (0.8¾ agar gelatin) in TBE buffer (0.089M Tris, 0.089M boric acid, 0.002M EDTA), electrophoresis is performed at a voltage of 100 volts. • 3. The electrophoretic colloid is stained with ethidium bromide '&quot; placed under a long-wavelength (300-360nm) UV lamp to determine the position of the desired DNA fragment, and the colloid together with DNA is cut off with a blade. 4. Put the colloid in an appropriate dialysis bag and fill it with 1 X ΓΒ E buffer solution to clamp the dialysis bag and clamp the two ends of the bag | to avoid the generation of air bubbles, lay it flat in the mud bath, and the electrophoresis solution in the tank must be Cover this bag too. 5. Electrophoresis at 100V for about 1, 5 ~ 2 hours. Look at the DNA under the UV lamp. If you have run out of the colloid and enter the solution in the bag, if you put it back in the electrophoresis tank, run it with the opposite electrode for about 2 minutes. 6. Carefully recover the liquid skull in the bag, and extract once with phenol and gas imitation. Add 0.1 volume of sodium acetate and equal amount of isopropyl (IPA), mix well, and place at -20 ° C for at least 2 hours. 7. Centrifuge at 4 ° C and 14000 rpra for 30 minutes, discard the supernatant, wash once with 70¾ and 100Γ alcohol respectively, and dry the DNA by vacuum centrifugation. 8: Add appropriate TE buffer to dissolve the dried DNA. 1. 2. 3. 2. Preparation of the probe: '1.2.3.2.1. Random mode selling method ("611 ^ 6" 8 &amp; ¥ 〇 口 6151 ^ 1 | 1,1983.): (Please read the back first Note for isC to fill out this page) • Install. -The paper size of the binding book is applicable to the Chinese National Standard Falcon (CNS) A4 specification (210X 297 mm) A7 B7 printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. /;) 1. Take 100 ~ 200ng of DNA, add sterilized deionized water to make the volume 5. Then heat it at 100 ° C for 3 minutes and quickly insert it into ice to make it cool rapidly. 2. Add ljitl BSA (10iUg / wl), 1.5ul hexamer (25ng / iil), lOwl 2.5x reaction buffer (0.5M Hepes, PH 6.6; 12.5mM MgCla; 25ntM / 3-propyl alcohol) ; 125 mM Tris, pH 8.0, 50 mM dATP, 50 mM dGTP, 50 mM dTTP), 5 u 1 [a-3 2 P] d CTP (1 0 nci / * 11, 1 M 1 K 1 e η ow enzyme) (BRL, 5 units / ill) mixed evenly. 3. Stand at room temperature and react for at least 2 hours. 4. Add TE buffer to increase the volume by 200ul. 5. Extract once with vinegar and acetone. Add 20m1 Ha0Ac, 3wl tRHA (20Mg / jal), 200m1 IPA, Mix well and place at -20 ° C for at least 2 hours.

6. Centrifuge at 14000 rpm for 20 minutes, aspirate the supernatant, and wash the precipitated probe DNA twice with 75¾ alcohol * and then dry the DNA by vacuum centrifugation. 7. Take 5 μl of the probe solution and measure its cpm using a scintillation counter to estimate the amount of probe solution required for hybridization. Probe solution volume U1) = 2 ~ 5xl05cpm / ml X volume of hybridization reaction solution (ml) + probe 1 volume of liquid radiation (cpm / μ 1) 1 .2. 3. 2. 2. In vitro transduction method (Me 1 ton et a 1., 19δ4): _ '1. This method uses pB luescr ipt H KS (+/-) as a carrier, and uses the paper standard to apply the national standard (CNS) 84 specification (210X 297mm) -16-III Pack IIIII Book-Line III (please read the notes on the back first and fill in this page) (printed by the Ministry of Economic Affairs Bureau of Central Standards Staff Consumer Cooperative A7 ______B7_ V. Description of Invention (//) It contains There is the persistence of T7 promoter and T3 promoter, add T7 RNA polymerase or T3 RNA polymerase to the test tube to make single-stranded anti-intentional RHA as a probe. In this experiment, Wang used this reaction to prepare the pending gene probe Needle (Tzou, 1990) to screen the promoter of the starch hydrolase gene of rice and determine whether the four starch hydrolases cDN A (RAMYC26, RAMYC27, RAMYC28, and RAMYC30) may be derived from our isolated α-starch hydrolase Genome DNA. 2. Take about 200 ~ 300ng of DNA, add DEPC HaO to make the volume 6 wi, then add Into 5itl 5x translation buffer, lwl 10mM rATP, 1 wl 10mM rCTP, U1 ΙΟιηΜ rGTP, 2m1 ImM r * UTP, 1 Ml. 0.75M DTT, U 1 RNase-b lock (40U / m1 RNas in), C2iil yeast t -RNA (1〇u1 / m1), 5 / ul a-32P_UTP (A mersham, 10 mci / ii 1), 1 M 1 T 7 or T 3 RNA polymerase, after mixing, react at 37 TC for 45 3. Add 20ul DEPC Η2〇, 5μ1 DNase I buffer (l〇x), 0.5mI DNase I (23u /; ul), after mixing well, react at 37 ° C for 15 minutes. 4. Same method 1 · 2.3.2. 1. Steps 5, 6, and 7. 1. 2. 3. 3. Probe hybridization: 1. Soak the dried NC filter paper in 5xSSC. 2. Transfer the NC filter paper to the device With an appropriate amount of prehybridization solution (50¾ formamide, —0.75M NaCl, 50mM Tris, pH7.5, 0.1% Na4P207, 1¾ SDS, ImM EDTA, lx Denhardt's solution, 0.1mg / ml 'salmon sperm DNA.) In a glass dish. Pre-hybridization solution (ml) = (0.04 The paper size is applicable to China ’s national standard falcon (CNS> A4 specifications (210X 297 mm) I n Pack III--order III-line (please read the notes on the back &quot;-/ (Fill in this page) 1. Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (/,) ml / cra2x area of each piece of NC filter paper (cm2) x number of NC filter paper>. Salmon sperm DNA needs to be heated to 100 ° C for 5 minutes, then quickly put it in ice to separate the DNA double strands before use. 3. Place in a 42 constant temperature box and shake for 3 hours. 4. Heat the probe containing the α-starch hydrolase gene It takes 3 minutes to 100 ° C (the RNA probe does not need to be heated), quickly insert it into the ice, wait for it to cool down, add it to the pre-hybridization solution, mix well, and put it back into the 42 t constant temperature box to react overnight 5. Discard the hybridization reaction solution, wash twice with washing solution (0.1xSSC, 0.1% SDS) at room temperature, and then shake and wash at 50 ° C for 30 minutes, measure with Geiger counter The level of radioactivity, if it is too high, wash with a washing liquid at 55 ° C or higher for 30 minutes. 6. Dry the NC filter paper, and then mix a small amount of (ct -35S) dATP ink on each of the four asymmetric points of the NC filter paper, so that the negative film can be used for reference for orientation identification after washing. 1. 2.4 Pure strain of starch hydrolase gene group Purification of: 1. Initially obtain the lysate with positive hybridization reaction from the previous steps, which contains the gene of α-starch hydrolase. Because the hybridization reaction map obtained from the exposed negative film may cover several plaque lysates, So these plaques need to be excavated to further determine which value is the real positive hybridization reaction, but the bacteriophage of these plaques ... The concentration may be too high * Therefore, serial dilution is necessary, and then select the appropriate concentration for probe hybridization ——- '2. After preliminary hybridization, use the tip of a pipette to dig out the ir contained in the positive reaction point of the hybridization to fill in this education)-The paper size of the binding and binding book is applicable to the Chinese National Standard (CNS> A4 specification (2H3X297mm ) 18 A7 ____B7_ printed by the Consumers ’Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. Description of the invention (/ stone) The plaque covered with lysate and 1 ml of plaque dilution (phage dilution buffer) (20 mM Tris-HCl, pH 7.5; 100 mM HaCl, 10 mM MgSO *), 20 / il gas imitate and mix thoroughly. 3. Let stand at room temperature for 2 hours to diffuse the bacteriophage out of the gelatin, averagely dissolving bacteria The plaques have about 10 8 ~ 107 phage particles. 4. Serial dilution method of phage: prepare 3 small 1.5m 1 centrifuge tubes, add 100wl, 180ul, 90a1 phage dilution buffer each, and then proceed to step 2 The phage stock solution is mixed with lwl and lOOwl phage dilution, which is 10-3 times the phage dilution solution, from 10 _ 3 times the solution to absorb 20u I and ISOu I dilution mixture, that is. Diluted 10_ 4 times, then It draws 10 and mixes with 90 * 11 of the diluent to make a dilution of 1 (Γ 5 times *), so 10-3, 10-4, and 10-3 phage dilution solutions can be obtained. 5. Take ΙΟΟίΙΙ of NM 538 bacterial solution (0D600 = 0.5 ~ 0.7) to 3 units. In a clean eppendorf test tube, then pipette 10-3, 10-4, and 10-5 times of phage dilutions of 100iil and The bacterial solution was mixed and incubated at 37 ° C for 15 minutes. 6. Take three 5ιβ1 test tubes and add 3ml of melted Top agar and 30wl 1M MgCl 2 respectively, and keep them in a 48TC water bath. 7. Mix the phage and bacteria mixture with Top agar evenly, and quickly pour it on the pre-warmed 37_3C LB solid medium (diameter 9cm) to spread it evenly. After Top agar has coagulated, incubate in a 37 ° C incubator Incubate overnight. '8 · Repeat steps 1. 2. 2., 1 · 2 · 3. And 1. 2 · 4. Until the medium contains, please read the notes on the back first h fill in this page) Pack. Order_ This paper size is applicable China National Standard (CNS) A4 specification (210X297mm) -19- ________B7_ V. Description of the invention (/ ^) The plaques of the α-starch hydrolase-based gene are reproduced only from a single plaque. H 'analysis of pure strains of rice α-starch hydrolase gene group: 2. 1. Purification of bacteriophage DNA: 1. Inoculate the host fine _ ^ »1 538 into lOral LB + Mg * 2 liquid medium (lOiiil per liter of LB) 1M MgS〇4), shaking culture at 37 ° C overnight. '2. Add 100ml of LB + Mg * 2 medium to 500ml Erlenmeyer flask, and then mix with 4ml of overnight host bacteria and 5 ~ 10® plaques from the same source (or 500m1 phage stock solution), shake at 37¾ Cultivate. Overnight. 3. The culture medium cultured overnight can see the corpses of bacteria eroded by the phage, so the culture medium is slightly clear, at this time, 5 / t 1 of DNase I (l〇ug / iil) and RNase (l〇 Mg / iil), and then incubated at 37- ° C with shaking for 30 minutes. 4. Centrifuge the Sorvall RC 5C centrifuge with a GSA rotor at 4 ° C and 8000rpin for 10 minutes. The upper clear solution is the phage solution. Printed by the Consumer Labor Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. In a Beckman centrifuge tube (Cat. No. 344058), add 6 ml cushion solution (40¾ glycerol, 20raM MgS〇4, 50mMNaCl, 10mM Tris-HCI, PH8.0 ), Then slowly inject the human phage solution until the injection is 0.5 c ai away from the nozzle. After equilibration, centrifuge for 1.5 hours at 27000rpm using a Beckma η Sw28 rotor, LB-M ultra-high-speed centrifuge. This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) -20-A7 B7 printed by the Negative Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (ΛΓ) 6. After centrifugation, discard the supernatant Then, take the centrifuge tube upside down and cut it with scissors near the bottom of the tube. Invert the centrifuge tube to avoid other debris contamination. Dissolve the precipitated phage in 1 1 NTE buffer (300 inM NaCl, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA), and then transfer it to an eppendorf test tube, and then deposit the remaining phage in 150 m 1 NTE buffer to precipitate once. -7. Add # amount of phenol to extract twice, and then extract once with chloroform. 8. Add 30μ1 4.4M ammonium acetate pH 5.2, 350wl isopropanol, mix well, and then the precipitation of phage DNA can be seen. Then, centrifuge at 1 4 0 0 0 p r m for 5 minutes at 4 ° C with an e p p e n d 0 r f centrifuge, that is, phagocytic and bacterial DNA can be obtained. 9. The precipitated phage DMA was washed once with 70¾ alcohol and then with 100Γ alcohol, and the DNA was dried by vacuum suction. 10. Add 40ml TE buffer (10mM Tris, ImM EDTA) to dissolve the dried DNA, take U1 DNA solution for restricted cleavage analysis. 2.2. Southern blot analysis: 1. After the effect of phage DNA with restriction enzymes, add a loading buffer of 0.15-fold (0.25¾: bromophenol blue, 0.25¾ Toluidine, 30 glycerol). 2. Use agar colloid (0.8% agar gel) in TBE buffer (0.089M

Tris, 0.089M boric acid, 0.002M EDTA), electrophoresis was carried out at 100 volts. 3. After electrophoresis, the agar colloid is stained with ethidium bromide, and the phase is illuminated under UV light. This paper scale is applicable to the Chinese National Standard (CNS) Α4 specification (210X 297mm) 21 IIIIJ-Pack II Order II n (please read the notes on the back first // r fill in this page) (Employee consumption of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 printed by the cooperative. V. Description of the invention (/ f) 4. The agar colloid is immersed in a denaturing solution at room temperature for about 1 hour, and then the neutralizing solution is used for 1 hour. 5. Bridge made of acrylic plate Cover the tray with a 3MM filter paper so that it can stretch under the bridge so that it can absorb the solution in the tray. Then cover it with a 3MM filter paper of the same size as the bridge, first on the filter paper Pour some lOxSSC solution to moisten the filter paper, and then put the colloid flat on the filter paper. The gene sieve membrane that has been wetted in lOxSSC in advance is flatly covered on the colloid to remove any air bubbles. Then cover it with two sheets to moisten with lOxSSC 3MM filter paper. Δ. Pour the appropriate amount of lOxSSC solution into the shallow dish, and cover the area around the bridge with the two layers of cleaning film, and then put another one on the filter paper of the mountain of colloid. Stack the absorbent paper, then top with the book Lightly press overnight. 7. Take out the gene sieve membrane and place it in an 80 Ό oven for 2 hours. 8. Put the baked gene sieve membrane in a hybridization bag and add 2xSSC solution to wet the gene sieve membrane. After squeezing out the excess 2xSSC solution, add an appropriate amount of prehybridization solution and shake the reaction at 42 C for 3 hours. 9. For the steps, please refer to the previous step 1.2.3.3 .. 2 · 3. Rice α-starch hydrolase gene Colonization of group DHA fragments: 2.3.1. Preparation of appropriate cells (E. coli XL 1-B strain): 1: Pick single colonies of XL1-B and inoculate in 5 ml of LB liquid medium * on 3 7 Cultivate overnight at ° C. 2. Pour 5m 1 of bacterial solution into 500m 1 of LB medium, shake at 37 3C to culture the paper. The paper scale is applicable to China National Standards (CNS) Λ4 specification (210Χ297mm) (please read the back first (Please fill out this page again).

* 1T 321685 A7 ______B7___ 5. Description of the invention (&gt; 〇) Raised to 0D55〇 = 0.35 ~ 0.5. 3. Centrifuge for 5 minutes at 4 ° C and 5000rF> m. 4. Discard the supernatant, place the centrifuge tube in a water bath, and dissolve the precipitated bacteria with 200 ml of ice-cold 0.1M MgCU. 5. Centrifuge at 5000 rpm for 5 minutes, dissolve with 200 ml of ice-cold 0.1M CaCla®, place in an ice bath for 20 minutes, and then centrifuge at 5000 rpm for 5 minutes. 6. Add 43ml of ice-cold 0.1M CaCla to dissolve the bacteria, then add 7ml of pre-chilled glycerin and mix well in the ice bath. 7. Separately pack the bacterial solution in Eppendorf test tubes and place in liquid nitrogen. Quickly freeze and store in -70 t refrigerator. 2.3.2. Conjugation reaction: 2.3.2.1 Dephosphorylation of linear DNA: 1. Using a single restriction enzyme, cut lOwg pBluescriptl KS, (+ /,) (Stratagene), and add TE buffer to make the volume Become 100ul. 2. According to the method of recovering DNA by agar colloid (steps 6, f, 8 in Section 1.2.1.1.), Purified linear DNA can be obtained again. Printed by Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 3. Add 20ul TE buffer to dissolve the DNA, then add 2 units of CIAP (calf intestinal alkaline phospholipase) and 2w I 10xC IAP dephosphorylation buffer (10mM ZnCI2 , 10mM MgCI2, 100mM Tris), incubated at 37T: 30 minutes. 4. Same as step 2. 5. Repeat steps 3 and 2 again. This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm). The A7 B7 is printed by the Employee Consumer Cooperative of the Central Bureau of Economic Development of the Ministry of Economic Affairs. 5. Description of the invention (&gt; /) 6. Add 20m 1 TE buffer to dissolve Phosphorylated plastids DNA, stored at 4 ° C. 2.3.2.2. Joining of DNA at the adhesive head: 1. Please refer to the method of recovering DNA from agar colloid (Section 1.2.3. 1. Steps 1, 2, 3). However, the preparation of the colloid was changed to Nusieve agar with a low melting point (FMC, # 10259), and 5 ul of ethyl bromide (10ug / wl) was added to each 100ial agar colloid. &quot; 2. On the ϋ V light, cut the colloid of DH A and place it in a water bath at 68 ° C for 10 minutes to melt the colloid and keep it in a water bath at 37 ° C. 3. Take 20.5W1 DNA, add 0.5ul of dish acidified plastid DNA (0.2 Mg / iil), 6 Du 1 5xT4 DNA ligase buffer (100 mM Tris, 25®M MgCl2, 25raM DTT, 250, ug / ul BSA) * 3wl T4 DNA ligase (1 unit / * il). 4. React overnight in a 16 ° C water bath. 2. 3. 2. 3. Conjugation of homogenous DNA: 1. Use agar colloid to recover DNA to obtain purified DNA at the end of the sticky end 〇2. Fail 40ul DNA, add 2m1 2mM dNTP mix, 3 / il Uenow enzyme (5n / wl), 5m1 l〇x fracture translation buffer (0.5M Tr is, 0.1M MgS〇4, 1mM DTT, 500iig / ml BSA), mix well, and react at room temperature for 30 minutes Make up the head _ head end. 3. Add 2ul 0.5M EDTA, and then extract once with phenol and chloroform, ^ / repeated agar_body recovery method of DNA (Section 1.2.3.1. The steps in this paper are applicable to Chinese national standards (CNS) A4 specification (210X297mm) 24 I --- ^ ----- 7 ~ install ------ order ------ line (please read the precautions on the back first P fill this page ) (321685 A7 B7 _ V. Description of the invention (&gt; xi 6 &gt; 7 &gt; 8) ° 4. Take 43.5til DNA, add 0.5m1 blunt end plastid DNA (0.2 wg / ul), 10wl 5x T4 DNA ligase buffer, 6 * il T4 DNA ligase (ul / ml). 5. Reaction at 16 t: overnight in water bath. 2.3.3. Transplantation of bacteria: • 1. Rice α-starch hydrolase gene After the group DNA fragment and plastid DNA were conjugated and reacted overnight, if it reacted in a low-melting point colloid, it was heated at 68 ° C for 5 minutes, and then incubated in a 37 ° C water bath. 2. Add 200jul of appropriate cells ( XU-Blue), placed in an ice bath for 30 minutes, 3 minutes. 3. Quickly put in a 42 ° C water bath for 3 minutes. 4. Add IOOmI LB medium, shake at 37 ° C for 30 minutes to 1 hour. 5 . Cultured in LB solids containing ampicillin (50 mg / ml) Each base is coated with 75ul X-sal (2%, 5-bromo-4-chloro-3-worm-yl- / 3-DH galactoside and l0wl IPTG (100mM isopropyl-; SD- Thio-galactopyranoside), and leave it on a sterile operating table for more than 15 minutes. 6. Spread the bacterial solution on LB solid medium containing X-Gal and IPTG and incubate at 37 ° C overnight. 2.4. Purify bacteria Plastid 0: 2.4. 1. _Small purification method: 1. Pick a single colony and cultivate it in a liquid of 5 m 1 LB + A mp (5 0 wg / Μ 1) — Culture based on, 37 1C shaking culture Overnight. This paper scale is applicable to the national standard (CNS) Α4 specification (210X 297mm) -25---------- "installed ----- ordered ----- line (Please read the precautions on the back first 〆fill this page) Ministry of Economic Affairs Central Standards Bureau Beigong Consumer Cooperative Printed A7 B7 Ministry of Economic Affairs Central Standards Bureau Beigong Consumer Cooperative Printed 5. Invention Description (&gt; Blade 2. Draw 1.51111 ® solution into an eppendorf test tube and centrifuge at 14000 rpm for 3 minutes at 4 ° C. 3. Aspirate the supernatant • Prepare ice-cold solution I (50mM glucose, lOraM EDTA, 25nM Tris-Cl, 4mg / ml) with 100ul Dissolve enzyme) to dissolve the precipitate Cells, mixed, reacted at room temperature for 5 minutes. • 4. Add 200ul of freshly prepared solution H (0.2M NaOH, 1% SDS) 'to mix well, and react in an ice bath for 10 minutes. 5. Add 150jil of ice-cold solution I (5M CHaCOOK, pH 4.8), mix well, and react in an ice bath for 10 minutes. 6. Centrifuge at 4 ° C and 14000 rpm for 10 minutes. 7. Aspirate the supernatant and extract once with phenol and aeroform, add equal amount of isopropanase, and place in -20 ° C refrigerator for at least 1 hour. 8. Centrifuge at 4 ° C and 14000 rpm for 30 minutes, discard the supernatant, wash once with 70¾ and 100¾ alcohol respectively, and dry the DNA by vacuum centrifugation. 9. Add 16.8wl TE buffer to dissolve DN). 10. This DNA is commonly used for DKA sequence analysis, then add 3.2 u 1 5M NaCl, 20m1 13¾ PEG (8000), mix and place in an ice bath for at least 30 minutes. 11. Repeat step 8 and take 16iil TE buffer to dissolve DNA and store at 4 ° C for D N A sequence analysis. 2. 4.2. Large-scale purification method: • 1. For 5 m 1 of the bacterial solution cultured overnight, add 2 50 m 1 containing ampicillin (5.0.1 on the back | matters (write this page) for installation.

, 1T line · This paper scale is applicable to China National Standard (CNS) Α4 specification (210 X 297 mm) A7 B7 5. Description of invention (&gt; printed by the Ministry of Economic Affairs, Central Standards Bureau, Beigong Consumer Cooperative) wg / ml) In 2 × ΥΤ medium (containing 16g of bacterial trypsin gland, 10g of yeast extract, 5g of NaCl, 0.5ml of ION HaOH) in 2 × ΥΤ medium, shake culture at 37 ° C overnight. 2. Centrifuge with GAS rotor at 5000 rpm for 5 minutes at 4 ° C. 3. Discard the supernatant, add 5 ml of solution I (25 mM Tris-Cl, 10 mM EDTA, 15% Sucrose, 2 mg / mU lysozyme) to dissolve the cells, transfer the cells to a 40 ml Oakridge test tube, place on ice React for 20 minutes in the bath. 4. Add 9.5ml of solution] I (0.2M NaOH, 1¾ SDS), gently shake the centrifuge tube up and down to mix evenly, and react in an ice bath for 10 minutes. 5. Add, add 7.5ral solution H (3M CHaCOONa, pH 4.8), mix gently and place in an ice bath for 20 minutes. 6. Centrifuge at 12000 rpm in SS34 rotor for 30 minutes. Filter the supernatant with two layers of gauze or one layer of mi rac loth, add an equal amount of isopropanol, and place in a -20 ° C refrigerator for at least 2 hours. 7. Centrifuge at 12000rpm for 30 minutes, discard the supernatant, wash the precipitated DNA once with 100¾ ethanol, and dry the DNA by vacuum suction. 8. Dissolve the DNA in 4.2ml TE buffer, add 5ul RHase (10 mg / ml) and let stand at 37 ° C for 20 minutes. 9. Add 4.5s CsC 1, gently shake to completely dissolve, then transfer the DNA solution to the Quick Seal tube (Beckman, 5.1ml, No. 344075) with a dropper. 10. Fill the centrifuge tube with ethidium bromide (nmg / inl), and after equilibration, seal the tube opening with Tub &lt; Sealer (Beckman). Please _ first read ―% Remarks &amp; Precautions Binding paper size is applicable to China National Standard (CNS) Α4 specifications (210 X 297 mm) 27 321685 Ministry of Economic Affairs Central Standards Bureau Bei Gong Consumer Cooperative Printed A7 B7 5 1. Description of the invention 11. Centrifuge with VTi 65.2 rotor at 20 ° C and 55000 rpm for 15 hours. 12 · Under a long-wavelength [JV lamp, use a needle to extract the plastid DNA, and then extract the red ethidium bromide with butanol several times until the DNA solution is clear and colorless. 13. Carry out dialysis in a dialysis bag in TE buffer for about 12 to 20 hours to remove CsC1, during which the TE buffer needs to be changed about three times. . 14. 'Collect DHA solution · Measure DfU concentration using a spectrophotometer. I. DNA sequence analysis The promoter of rice α-starch hydrolase gene: 3, 1. The 5 'end of the α-starch hydrolase gene with progressively shortened sequence (Henikoff, S., 1984) 1. The principle of this experiment is Two restriction enzymes are used to cut two different parts of DNA, one part has a protruding 3 'end, and the other part has a protruding 5' end or blunt end. * External nuclease Π [(ΕχοΒΙ) The principle of specific cutting action at the protruding 5 'end or flush head end. Taking samples after different reaction times can obtain a series of gradually shortened D Ν Α. 2. Take lSjug DHA to act with restriction enzymes Bam HI and Bstx I at 37 ℃ for 2 hours. 3. The method of recovering DNA by repeating the agar colloid (Section 1. 2. 3. Steps 6, 7, and 8 Bu 4. Dissolve the DNA in 1 H2〇, and add 2 mL of 10 mL Εχο Μ buffer (660 mM Tris pH · 8 · 0, 6.6mM MgCl2.), Keep in a water bath at 30 ° C. The paper size is applicable to China National Standard (CNS) A4 specification (210X 297mm) A7 -------- -B7 _____ 5. Description of the invention (&gt;ό;) 5. Preparation of S 1 m · 〖X (5 9 · 5 «1 彳: 〇, 9.5 u 1 7.4 XS 1 buffer, 5 〇unit S 1 nuclease), 7.4 xs 1 buffer (0.3 MCH 3 C Ο Ο K, Ρ Η 4.6, 2.5 Μ M a C 1, 1 〇m Η Z n S Ο 4, 5, Ο% glycerol ) 6. Take 7.5 u 1 S 1 mi χ and aliquot it into 8th e f> ρ e η d ο rf test tubes and keep them in an ice bath. 7. Add 400 units of DNA sample in step 4 Exo H [enzyme's rapid mixing, placed in 30 t: reaction in a water bath :, 8. Take out 2.5 u of the anti-solution S 1 mi '&lt; thoroughly mixed in an ice bath every one minute and place in the chamber The reaction was carried out at a temperature of 30 minutes. &Quot; 9. Add 1 u 1 S 1 stop buffer (0.3 M T risbase, 0 〇5 Μ EDTA), after mixing, heated at 70 t: 10 minutes. 1 〇. Take 2m 1 of the DNA samples at each reaction time point for electrophoresis analysis of agar colloids. 11. Add 1.5mI Klenow mix (15m1 lxKlenow buffer, 5 units of Klenow enzyme), incubate at 37 ° C for 3 minutes, then add U1 dNTP mix (dATP, dCTP, dGTP and dTTP each 0.125 m Μ), and react at least 5 at 37 ° C 12. Add 40m1 zymase mix (395iM H20, 50ul l〇x zymase, Ministry of Economic Affairs Central Standards Bureau employee consumer cooperatives printed buffer, 50jil 50% PEG, 500mM DTT, 5 units of T 4 DNA ligase) At room temperature, it should be at least 1 hour. 1 3. Repeat steps 2, 3, 3. 3 on bacterial colonization steps 2, 3, 4, 5, 6 ° 3.2 DNA sequence analysis: …-Using S anger's dideoxynucleic acid bond termination method, using the Chinese standard standard falcon (CNS) Λ4 specification 210 X297 public frame at the standard of S equenase paper. A7 printed by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs B7 5. Description of the invention (&lt;) kit (USB, Version 2.0, No. 70496), synthesis Unit DHA, and using (α - 3 5 S) d ATP calibrated nucleotides. 3.2.1. Preparation of colloid: 1. Clean the glass sheet with detergent and wipe with 95¾ alcohol. Once dry, the inner surface of the upper glass sheet is evenly rubbed once with 5¾ dichlorodimethylsilane to ensure its Smooth, and then combine the glass sheet with Spacer (refer to Maniatis et al, 1989, Figure 13.8) ° '2.' Take 48ml Urea mix (420g per liter containing urea, ΙΟχΤΒΕ 100 ml), 36ml of acetamide stock solution (per liter Acetamide 193g, urea 420g, bis-propionamide 6.7g, 10χ ΤΒΕ ΙΟΟπιΙ) and 600 / ϋ 10¾ persulfate, mixed evenly and placed in an ice bath. 3. Take α〇ηι1 mixture and add 20ml TEMED (Ν, Ν, Ν, ϋ, -tetra-methyl vinylamine), after mixing, use the pipetteman tip to quickly edge the spacer between the glass pieces, and then clamp it with a clip Tighten the periphery of the glass Η and let stand for about half an hour. 4. Add 57.5iil TEMED to the mixed solution of acrylamide and mix well. The prepared colloidal model of glass flakes was slanted at an angle of 45 ° to the horizontal at the bottom, and pipet-aid was used to suck the mixture of acrylamide with a 25ml pipette, and slowly inject it from the lower bevel. 5. After filling, the glass sheet is slowly laid down in a flat lying manner, and the top of the top is styrofoam plate so that the top is about 5 cm higher than the bottom, and then the flush end of Shark's tooth comb is inserted into the top from the top 0.5 cm. 6. Clamp the top with a clamp and let it sit for more than 2 hours. If you want to put it overnight ~ ~, seal the top with a cleaning film to prevent the gel from drying. (Please read the precautions on the back-T &quot;% to write this page) Installed-Line Tai Mi Chu Oilfield Middle Group Dang Ji Ji (CMS) A4 specification (210X297 public yak) 2 0 Printed by the Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economy System A7 _B7_ _ V. Description of the invention (&gt; f) 3.2.2. Double strand DNA sequence analysis:

1. Take 16ul of supercoiled double-stranded DNA (Ug / μl) and add 4ul of 2M NaOH, mix well and react at room temperature for 5 minutes. 2. Add U1 mold vendor (l〇ng / ul), 6m1 3M CHaCOONa and 80ul 100¾ alcohol, mix and place in -20 t: refrigerator for at least 2 hours. 3. Centrifuge at 14000 rpm for 30 minutes. The precipitated DNA is once in 70% and 100% alcohol, and the DNA is dried by vacuum centrifugation. 4. Take 4 eppendoff test tubes, add 2.5m1 ddATP, ddGTP, ddTTP respectively, and place them in a 37 ° C water bath for pre-warming. 5. Take 8 (· l1 HzO to dissolve DNA, then add 20 «1 Sequenase reaction buffer, U1 DTT, 2m1 diluted mix (0.4U dGTP logo ati ^ + 1.6« 1 ΗίΟ + 1 μ 1 (α-3 5 S ) d ATP (10iici / wl), 2m1 diluted sequenase (0.3m1 Sequenase + 1.7wl enzyme dilution buffer), react at room temperature for 5 minutes. 6. Take 3.5a 1 of each radioactively labeled DNA reaction solution and pre Warm 2.5 / tl ddATP, ddGTP, ddCTP, ddTTP are fully mixed and reacted for 5 minutes at 37 ° C. 7. Add 4 «1 stop solution to each tube, mix evenly and heat at 86 ° C for 5 minutes before placing on ice In the bath, electrophoresis analysis can be carried out after cooling. 3.2.3. Single strand DNA sequence analysis: The principle of this test is to use pBI rescript H KS (+ /-) plastid to replicate f I (+ /-) phage Starting point, when the plastid replicates, infection via bacteriophage-positive strand [SK (+), including /?-Galactosidase gene translation IIIIII loading IIII-. Line (please first Read the precautions on the back c-% to write this page) (This paper size is applicable to China National Standard (CNS) A4 specification (210X297mm) 31 -X 1 Central Ministry of Economic Affairs 321685 A7 _____B7 printed by Beigong Consumer Cooperative of the Bureau of Standards V. Description of Invention (&gt; f) Cryptographic Unit] and negative shares [US (-), including non-deciphered cryptographic units] can be integrated into the bacteriophage gene group. Therefore Purify the DNA of the bacteriophage, and use the template in different directions (KS or SK template) to perform two-way single-strand DHA sequence analysis. 1. Pick a single colony and add it to the liquid medium of 2xYT + ampicillin (75ug / ml). 2. Add 108 ~ 103pfu / ml of bacterium VCS-M13 (Stratagene, Ho. 200251) or R408 (Stratagene, No. 200252) * Shake culture at 3710 for 2 hours. 3. Add card to the medium of VCS-M13 Nahuisu (70ttg / W 丨), but the medium of R4; 08 is not added, shaking culture at 37 ° C for 10 ~ 12 hours 0 ^ 4. Take 1.5ml of bacterial solution and centrifuge at 14000 "pm for 5 minutes. 5 Aspirate the ltnl supernatant, add 150m 1 PS buffer (20¾ PEG, .2.5M NaCl), mix and place in an ice bath for 30 minutes. 6. Centrifuge at 14000rpm for 20 minutes, aspirate the supernatant, 400m1 SE buffer (0.3M CH3C00Na, 1mM EDTA) dissolves the precipitated phage particles. 7. Extract once with phenol and chloroform, add equal amount of isopropanol, and place at -2 0 ° C for about 1 hour. 8. Centrifuge at 14000 rpni for 30 minutes, aspirate the supernatant, wash the precipitated DNA with 70¾ and 100% alcohol each time, and dry the DNA by vacuum centrifugation. O: 9. Take 56ul HzO to dissolve the DNA and add it to the U 丨 template. luI DMSO, 2ul

IJI (please read the notes f% on the back to write this page)

、 1T line paper size is applicable to China National Standard Falcon (CNS) A4 specification (2 丨 0 X 297 mm) 32 Printed A7 _____B7___ by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economy V. Description of invention Sequenase reaction buffer, mixed Evenly. 10. The remaining steps are the same as the steps 4, 5, 6, and 7 of the double-strand DNA sequence analysis. But in step 4, each tube needs to add 0.25 / 11 DMS0. 3.2.4. Computer analysis of DNA sequence: Computer analysis of DNA sequence was performed by GCG sequence analysis computer software (the Genetics Computer Group, University of Wisconsin, Version 5.0, June 1987) (Devereux et al., 1984). &lt; W. Transplantation of rice suspension culture cells-using the electro-penetration method: 4.1. Rice suspension culture · 1. In a 125ml Erlenmeyer flask, 25ml of MS (Appendix I) liquid medium containing 25% sucrose is added and added. .5m 丨 suspended rice (Tainong No. 67) fine monument (about 0.2 g). 2. Incubate at 25 ° C, 12 hours light and 12 hours darkness at 120 rpm with shaking. 3. Subculture every 7 days. 4.2. Transplant the gene into rice cells using the electric penetration method. 1. Take the suspension culture cells of rice grown on the third day, absorb the culture liquid in the triangular flask, wash it with the washing medium (Appendix II) CPW7.4. Wash it once, move the fine roar into the culture dish, and add 5 m I enzyme solution (Every 100 ml of CPW7.4 contains 4% cellulase RS, 1¾ macerozyme R-10) Reaction at room temperature for 30 minutes. 2. Collect the enzyme-treated cell solution, transfer it into a 15 ml centrifuge tube and centrifuge at 800 rpm for 3 minutes. -&quot; '3. Remove the supernatant and wash the pelleted cells twice with CP W7.4. After that, the paper size is applicable to China National Standard Falcon (CNS) A4 specification (210X 297mm) -33-(please first Read the precautions on the back (4) and write this page). Packing. 321685 A7 B7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economics. 5. Description of the invention ($,) EPR buffer (10¾¾ glucose, 4inM CaCU, 10mM Hepes, p Η 7. 2) Wash once. 4. Add 5ιπ 1 EPR buffer to suspend the washed cells, and then add 20 α-starch hydrolase gene promoter-GUS gene fusion vector (PHHE132, ΡΕΕ132, etc.) and 10ug / ml The salmon sperm DNA was mixed evenly and incubated at room temperature for 1 hour. 5. 'Take 0.8ml of the cell suspension into the Gene Pulser cuvette with a 0.4cm electrode gap and let it stand in an ice bath for 15 minutes. 6. Place the cuvette in Gene Pulser (BIO-RAD), set the voltage: (Vo) to 150V, capacitance (Co) to 960uF, resistance (Ω) to 400 ohms, and shock time to 120msec. After the setting is completed, electric shock is applied to transfer the DNA into the cells. 7. Place the shocked cells in an ice bath for 15 minutes. 8. Transfer the cells to a Petri dish with 24 cells (Costar, No. 3424) +, add 3ml of KPR (Appendix III) liquid medium, seal the periphery of the Petri dish with paraffin film, and culture at 50 rpm in the dark at room temperature 3 husband. 4.3. GUS assay: 4.3.1 Preparation of 1 ^ color reaction solution (7-8 丨 11 reaction solution): 1. Dissolve 5mg X-glu in 100ttl DMS0. 2. Add 10 ml of reaction buffer (10 mM Na ^ EDTA, 100 mM NaHaPCU.HsO, 0.1% Triton, pH 7.0). '3. After mixing, each K5〇iii is packed in eppendorf test tubes' (please read the precautions on the back P% to write this page). Packing. The paper size of the binding book is applicable to the Chinese National Standard (CNS) A4 specification (210X297 Mm) -34- V. Description of invention A7 B7 Printed and stored in the -20TC refrigerator by the Employees Consumer Cooperative of the Central Bureau of Prospects of the Ministry of Economic Affairs. 4.3.2. GUS analysis: 1. After culturing the cells for 3 days, remove the culture solution, add 50 μl of X-glu reaction solution, and let it stand in the dark in the greenhouse to react for more than 3 hours. 2. Remove the cells, in Observe the staining of cells under a microscope.藠 詢 结 結 '1. Analysis of pure rice ot-starch hydrolase gene group DNA strains: using rice ct-starch hydrolase cDNA pure strains and RAMYC30 coding house as probes to screen the rice gene group DMA repository, A total of 16 pure strains of gene groups were obtained. The restriction enzyme "Sal I" was used to separate the rice genomic DNA from the bacteriophage DNA k, and it can be seen from the electropherograms (eg, 1A, 1C, 2E) that these genomic DNAs were cut into several segments. Among them, the electropherograms of RAMYG28 and RAMYG18 are the same, which may be the same pure strain. RAMYG17 and RAMYG19 may also be the same pure strain. 0 In order to determine what is obtained in the DMA repository of the obtained gene group, it is true that it contains rice α-starch hydrolase For pure strains of genes, after separating the DNA of Figures 1A, 1C, and 2E by colloid electrophoresis, transfer the DNA to the gene sieving membrane by Southern blotting, and then combine it with the RAMYC30 coding region or 5 'of the RAMYC28 coding region. The probes made at the end were hybridized, and the results (Figure IB, ID, 2F) showed that RAMYG10, RAMYG11, RAMYG14, RAMYG20 did not have a positive reaction, so it is inferred that these 4 pure strains may not contain the ct-starch hydrolase gene A total of 12 pure strains of α-starch hydrolase gene group were obtained. Figures IB, ID, and 2F show that some pure strains have a 2 to 5 painting hybridization positive reaction, (please read the precautions on the back to write this page) • The paper size of the binding and binding book is applicable to China National Standard (CNS) Α4 specifications (210X297mm) 35 B7_ V. Description of the invention (») It is speculated that the α-starch hydrolase gene contained in these pure strains may be divided into several segments by Sa 1 I, or that the same DH A contains more than one α- Starch hydrolase gene (Huang et al, 1990a). A ·· Identify the 5 'end of the α-starch hydrolase gene: The 5' and 3 'end probes used in this experiment are all DNA probes prepared by in vitro methods. This probe contains a Small plastid [pBI lies crip t &quot; H KS (+ /-)] multiple implanted DNA fragments, this section of DNA may have high homology with 1.6kb and 0.5kb in the label (lkb ladder, BRL) 0 'sequence, so there is a strong positive cross-reaction in the places marked 1.61 ^ and 0.51 ^. In order to determine the 5' end of the α-starch hydrolase gene of the pure strains of the selected rice gene group, In order to isolate the promoter site, the in vitro translation method was used to obtain the 5 'ends of the coding regions of RAMYC28 and RAMYC30, with lengths of 320b and 430b, respectively, plus the 3' end of HS501, with a length of 350b (Yu et al, 1990) Three kinds of DNA probes were made. Through the results of molecular hybridization (Figure 2B, 2D, 2F, 2H), it was found that RAMYG6, RAMYG8, RAMYG17, 1TAMYG18, RAMYG21 and RAMYG28 all have positive hybridization reactions. Printed by Beigong Consumer Cooperation, these pure strains may contain alpha- The promoter part of the powder hydrolase gene. Other pure strains may also contain the 5 'end of some α-starch hydrolase genes, but may have low homology to the DNA sequence of the probe used in this experiment, so there is no positive reaction. . In the two pure strains of RAMYG6 and RAMYG28, there are two DNA fragments showing _ • are now reacting (Figure 2F), so it is inferred that the probe used in the experiment (RAMYC28- This paper scale is applicable to the Chinese National Standard (CNS) Α4 Specifications (210x 297 mm) A7 B7_5, the invention of the consumer cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs of the Ministry of Economic Affairs, the invention description (&gt; MO Ο 5 ·), in which the DNA sequence may be between the junction of the two DNA fragments or the second segment The DNA fragments are all highly similar in composition. Later experiments (Figures 11 and 12) confirmed that the DNA sequence of RAMYC28-5 'did have a high degree of homology with the DMA sequences of RAMYG28a and RAMYG6c. As for the RAMYG28b and RAMYG6a The weaker positive reaction may be due to the low degree of homology between the probe's DNA sequence and the two pieces of DNA. B. Determine whether the known &amp; ^ α-starch hydrolase cDNA may have been derived from Genomic DNA: use The α-starch hydrolase gene (RAMYC26, 27, 28, 30). The 3 'end is a gene-specific probe (gene-specific probe) for molecular hybridization to understand the four possible sources of cx-starch hydrolase cDNA From the isolated genome DNAs, the hybridization results (Figure 3B, 3D, 3F, 3G) show that RAMYC26 is a pure cDNA strain derived from RAMYG8, while RAMYC27 and RAMYC28 are pure cDNA derived from RAMYG17 and RAMYG28, respectively. Strain (please refer to Table 1). The results show that RAMYG6, RAMYG18 and RAMYG28 may be the gene group DNA containing RAMYC28, while RAMYG18 and RAMYG28 may be the same gene; RAMfGS may contain the gene group DNA of RAMYC26; RAMYG21, RAMYG22 and RAMYG23 may contain the gene group DHA of RAMYC27. In Fig. 3F, RAMYG18 also has a positive hybridization reaction with a held-in gene probe at the 3 'end of RAMYC27. It is possible that the 3' end of RAMYG18 and RAMYC27 also have a similar composition of DN A sequences to some extent, so they also exhibit a positive hybridization reaction. 3. Sub-transplantation of DHA fragments of rice α-starch hydrolase gene group ~ _ The pure strains of genomic DNA and α-screened from the rice genomic DNA library-(Please read the notes on the back to write this page)- · The paper size of the binding book is applicable to the Xinguo National Standard (〇 ^) 84 specifications (2〗 0/297 mm) 37 321685 A7 B7 The coding area of the starch hydrolase gene printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs The probes at the 5 'end and the 3' end were hybridized DNA fragments (Figure 5), part of which were transferred into pBluescript 11 (5 (+ /-) vector, as shown in Figure 6. Among them 1? / ^ ¥ 01732 (Figure 4 dagger) and RAMYG28a2 DNA fragments cut by restriction enzyme cleavage, and then transfected into pBluescript vector. Fourth, the DNA sequence analysis of the 5 ′ end of the rice ct-starch hydrolase gene: (A) DNA sequence analysis of the 5 'end of the RAMYG17' gene: The probe made at the 5 'end of the coding region of RAMYC30 has a positive hybridization reaction with 1? 4 »^ 01731 (Figure 2b), so 1 ^^^ 61731 may be Promoter site containing α-starch hydrolase gene. Using restriction Sal I and EcoRI can reduce 4.5kb RAMYGlJa! It is 1.2kb of RAMYG17a2 (Figure 4), and it can still react with the 5 'end of RAMYC30, indicating that RAMYG17a2 may still contain the promoter site of the α-starch hydrolase gene. The deletion of RAMY 01732 by Exo SI 1) ^^ Fragments are gradually shortened, and then the DNA sequence is analyzed by the Sanger double deoxyribonucleic acid bond termination method. The analyzed DNA sequence is then compared with the published rice O (-starch hydrolase gene using GCG system. RAMYG17 a2 Although the DNA sequence is only 1200bp, it has been found to be identical to the pure λ 0Sg2 strain (Huang eta 1., 1990) (Figures 7 and 8), which means that the RAMYG17 33 and the pure λ 0Sg2 strain should belong to the same gene According to published reports, the signal peptide site of the α-starch hydrolase gene can be found (as shown in Figure 8), and there is a brain EcoN I restriction enzyme on the DN Α sequence immediately after the signal peptide site The cutable part, so use EcoNI to cut at the 5 'end of 1? / \ 14th generation 1731 * This can cut the 5' end of different lengths containing different lengths of the cc-starch hydrolase gene promoter and please read the back Notes on binding book The paper scale is applicable to the Chinese National Standard (CNS) Α4 specification (210X297mm). The consumer consumption cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs has printed the A7 _____B7_V. Invention description (#) The 3 'end contains the DH A fragment of the signal win chain. Figure 9) These DNA fragments can be connected to genes that want to express proteins in the future to construct a protein expression vector and secrete bamboo proteins out of cells by the function of signal peptide chain. (B) DNA sequence analysis of the 5 'end of the RAMYG6 and RAMYG28 genes: The probes made at the 5' end of the coding region of RAMYC28 and the RAMYG6 and RAMY G28 were hybridized with positive reactions, and the DNA fragments with positive reactions were named as RAMYG6a, RAMYG6c, RAMYG28a and RAMYG28M (Figure 2F). After DNA sequence analysis, it was found that both RAMYG28a and RAMYG6b contained the 5 'promoter site of the α-starch hydrolysis gene. RAMYG6b contains a 5 'promoter region that has no homology with the 5' end of the coding regions of RAMYC2 and 8. RAMYG6C does. There is no positive reaction between the former and the dish needle, and the latter does (Figure 10). During the experiment, Huang et al. Published the DNA sequences of two α-starch hydrolase genes, which they named RAmy3D and RAmy3E. After comparing the DNA sequences, we found that RAMYG28 and RAmy3E should be the same pure strain (Figure 10, 11), while RAMYG6 spans the 3 'end of RAmy3E and almost covers the entire RAmy3D gene (Figure 10, 11, 12). The research by Huang et al. (Huang et al., 1990) has pointed out that RAmy3D (= RAMYG6) and RAmy3E (= RAMYG28) are located on the same chromosome, and the distance between the two is about 4. Okb (Figure 10). DNA sequence analysis also confirmed this result. V. Construction of GUS expression vector: A. Construction of pBSI, pBXE and pBSfll (presented by Divid McEU "y") "pBI 101.1, pBI 101.2, pBI 101.3 (CLONTECH, No. Please read the notes on the back first" ^ 'Fill in this page)-Pack. The size of the line paper is applicable to the Chinese National Standard (CNS) A4 (210X297 mm). The selected or not printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs is based on common causes. Breaking tool-My I judged the species to be difficult to establish, and the change was due to the change. The neutrophilic fine plants were planted to the current plant. The number of the record was A7 _B7_ V. Invention description (&gt;;;) 6022) The E. coli GUS gene (2 kb) pseudo-deficient in the promoter was transfected on the polylinker of the Agrobacrteri um t umifaciens binary plastid vector pBIM19, and then the blue eosin (nopaline) of the Agrobacterium Asrobacter ium T plastid ) The terminator (260bp) of the synthase (NOS) gene is connected to the 3 'end of the GUS gene. Therefore, the promoter of the plant gene can be easily inserted into the polylinker at the 5' end of the GUS gene. Promoter Drive the transcription of the GUS gene, and can be engaged in the study of gene regulation. Because the replication efficiency of plastid pB IN 19 in bacterial skulls is very low and it is not easy to purify in large quantities, the restriction enzymes Bam HI and spel were used to separate pBIIOI. 1, pBI 101.2 and The 01 ^ gene contained in 98101.3 and the 0 terminator were isolated from the? 811 &lt; 19 vector, and the 0 "fragments of PBI 101.1 and pB 101.3 were inserted into pBluescritIKS (+), a plastid cut with restriction enzymes and spel, respectively Plastids PBS I and pBS I were obtained. The DNA fragment of pBI101.2 was inserted into the plastid pBlusecript I KS (+) cut with restriction enzymes BamHI and XbaI to obtain pBX E (plaque 13A). The difference between pBSI, pBXI, and pBSII lies in the 5 'end of the GUS gene, and the reading frame has a nucleotide difference. Therefore, the plant promoter can choose the appropriate reading frame to insert and construct a GUS expression vector . B. Transplant the GUS gene to a plastid with the gene of the alternative symbol-anti-moisture emblem because PBS I, pBX H, pBS III (Figure 13A) do not have an alternative paper size applicable China National Standard (CNS) Α4 specification (210X297mm) I installed I; swallowed. I line (please read the notes on the back ^ Λ fill in this page) (printed by the Ministry of Economic Affairs Central Standards Bureau employee consumer cooperatives A 7 B7 5 2. Description of the invention (y) The marked plastids are very important. The GUS genes in pBS I, ρΒΧ ϋ and PBs ΠΙ, together with the T 7 template and T 3 template and the multiple implanted sites between them are taken from the pBluescript vector using the restricted PVU Π Isolated from the medium (Figure 138), and then inserted into 1 ^ 1 ^ 11 [cut? 11 ^ 132 (Figure 13C) (Hayashimoto et al, 1990) vector, the result was pBX H-132 and pBSIB-132 (Figure 13D) , The GUS gene transmission direction of this diploid is the same as that of the moisture resistance gene (Hph), while the transcription direction of the GUS gene of pBS 1-132 (Figure 13E); the transcription of PU and moisture resistance gene In the opposite direction, a total of three plastids with GUS and anti-moisture emblem genes were obtained, and their DNA lengths were 7.Okb. The plastid can provide functions for analyzing various gene promoter functions in the future. C. Constructing a fusion vector of the rice α-starch hydrolase gene promoter and the GUS gene 1. Constructing a fusion vector of the RAMYG17 promoter and the GUS gene. The restriction enzyme map and graphic description of RAMYG17ai have obtained three DNA fragments containing pH- (0.88kb), PAE (1.7kb) and pSE (2.4kb) containing the promoter of α-starch hydrolase gene. Insert pBS I cut with restriction enzyme Snia I (please refer to the description of materials and methods for the joining method). According to the principle of gene triple code, decode from the starting point of protein translation ATG of pHE, pAE and pSE * until the GUS gene The translation start point of ATG must conform to the reading structure of the triple code. The promoter-GUS fusion vectors that have been joined are named pHE-Ι, pAE-Ι and pSE-Ι (please refer to Figure 14A, 14B, 14C Bu 'experiment At this point, the laboratory obtained plastid pTR A 1 32 (the Chinese standard (CNS> A4 specification (210X297 mm) is applicable to the paper standard 41 III n III —5 -n ^ II n ϋ I n IIIIII ^ (please Read the notes on the back first i- ?. Fill in this page) "A7 B7 by the consumer cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs V. Presented by Up Hayashimoto) · The use of restriction enzymes ρνιιΠ in pHE-I, pAE-I and pSE-I The DNA fragment of the promoter-GUS gene, together with the T7 promoter and the T3 promoter and the multiple implantation sites were separated from the pBluescript vector (Figure 14C), and then inserted into the pTR A 132 vector cut with Hindi (Figure 14 D) , The results obtained pSE 132 (11.4 kb), pAE 132 (9.7 kb) and pHE 132 (7.9 kb) three kinds of fusion vectors with anti-moisture gene (圔 14E). Among them, the translation direction of the GUS gene of pSE 132 and pAE 132 is the same as the translation direction of the anti-moisture gene, but the translation direction of the GlfS gene of PHE 132 is opposite to that of the anti-moisture gene. 2. Construct the fusion vectors of RAMYG6, RAMYG28 promoter and GUS gene. The RAMYG6 and RAMYG28 gene promoters are located in the DNA fragments of RAMYG6b and RAMY G28a25, respectively (Figure 10). EcoNI and Sal I can be used to hydrolyze the α- 'starch of RAMY G6b. The DNA sequence of the enzyme gene 5'end and the signal peptide chain (total length about 1.6kb) was isolated from the plastid pBluescript. However, RAMYG28aa cannot use restriction enzymes to separate the 5 'end of the α-starch hydrolase gene and the DN A sequence of the signal win chain, because there is no suitable restriction enzyme available at the end of the DN A sequence of the signal win peptide chain. Therefore, using the method of Exo Μ nuclease removal, the DNA was gradually shortened from the V-end of RAMYG28a2 until it was near the signal peptide DNA sequence (Figure 11, thick arrow) and then the α-starch hydrolase was restricted with the restriction enzymes Apal and EcoRV The 5 ”end of the gene and the DMA sequence of the signal peptide chain are separated from the plastid PB | lies crip t. Therefore, according to the principle of the gene triple code, the two segments of DN containing the promoter and signal peptide bond separated by RAMYG6b and RAMYG28az A fragment was inserted into the plastid pBS _ dish-132 cut with restriction enzyme Η ί nd I to obtain RAMYG6-132 (8.61cb) (Figure 15) and RAMYG28-132. Please read the notes on the back ^ -s -install- -ΛFill in this page) The paper size of the binding book is in accordance with Chinese National Standard (CNS) Α4 specifications (210X297mm) Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 321685 at _ B7 V. Description of Invention (Mod P) (I) (8.2kb) (Figure 16) the fusion vector of cx-starch hydrolase gene promoter and GUS gene. RAMYG6-132 and RAMYG28-132U) The translation direction of the GUS gene is the same as the translation direction of the anti-moisture gene. DNA sequence and GOS gene of the signal peptide chain However, there is a terminating translation signal TGA (Figure 15D, 18D). In order to make the constructed fusion vector between the DNA sequence of the α-starch hydrolase gene's signal It chain and the GUS gene, there is no termination. Translate the code, and then insert the restriction enzymes Apal and EcoRV from the 5 'end of the alpha-amylase gene isolated from RAMYG28a2 and the DN A sequence of the signal peptide peptide into the plasmid PBSI-132 cut with the restriction enzyme clal to obtain RAMYG28-132 U) (Figure 17C) ^ The translation direction of the GUS gene is opposite to the translation direction of the hygromycin-resistant gene, and there is no termination of the translation code between the DNA sequence of the signal wins chain and the GUS gene (Figure 1 7 D) 6. Transplant the GUS expression vector into rice suspension culture cells using the electro-penetration method. Using the electro-penetration method, three GUS surface mirror vectors, such as PHE132, PAE132 and RAMYG6-132, have been transformed into rice Suspension cultured cells. After 3 days, the reaction was colored with Xg I u, and the transient performance of the GUS expression vector was measured. The results showed that the pHE 132, PAE132 and RAMYG6-132 and other three GUS expression vectors had blue cells. Performance (Figure 18B, 18C) In contrast, the cells in the control group were transformed with a promoter containing no cc-starch hydrolase gene and a vector containing only the GUS gene. After the same treatment, the cells were not colored (Figure 18A). From this &gt; · Prove that PHE132, PAE132 and RAMYG6-132 can be normal in rice cells. This paper scale is applicable to China National Standard (CNS) A4 specification (210X297mm) -43-I i IIIIIII Pack III Order __ II &quot; Line (please read the precautions on the back first and fill in this page) ^ ______B7 5. Description of the invention (what: /) performance. Whether PSE132, RAMYG23-132 (I) and RAMYG23-132 (I) can also perform normally in the cell, need to be confirmed by further experiments. ^ * The application of gene transfer technology to study the expression and function of eukaryotic genes to promote the understanding of the regulatory mechanism of higher biological genes. It is known that sugar-deficient treatment is applied to the culture medium of rice suspension culture cells, and certain α-starch hydrolase genes in the cells will be expressed in large quantities, and α-starch hydrolase is also produced and secreted in large amounts into the extracellular medium (Kuo, 1990). This phenomenon of massive expression of the α-starch hydrolase gene induced by sugar deficiency, in the end how cells transmit the signal of sugar deficiency, how to sense the signal of sugar deficiency and regulate the large expression of the retained α-starch hydrolase gene, and will α-starch hydrolase is secreted out of the cell. This is the subject of our interest. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. This case has screened 12 different strains of pure α-starch hydrolase gene DNA from the rice genome DNA repository. This was further confirmed by probe hybridization and DNA sequence analysis. At least 4 out of 12 pure strains of the α-starch hydrolase gene group contain the DNA sequence of the 5 ′ end of the α-starch hydrolase gene and the signal peptide chain. The four pure strains are RAMYG6, RAMYG8, RAMY G17 and RAMYG28. It is worth noting that after the hybridization of the probes made by RAMYG8 with the coding region of RAMYC30 (Tzou, 1990), five DNA fragments showed a positive reaction (Figure 1B), and the total length of the five DNA fragments was about 10 kb * According to the results of DHA sequence analysis of RAMYG8C (Lo, personal contact), RAMY C26 (Louou, 1990) is a pure cDNA strain derived from RAMYG8, and RAMYG8c contains the DNA of the 5 ′ end of the RAMYC26 gene and the signal peptide bond sequence. Figure 3B / shows the specific gene probes made at the 3 'end of RAMYG8b and RAMYC26. The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm). V. Description of the invention (A »B7 Economy The Ministry of Central Standards Bureau employee consumer cooperative printed a strong hybrid positive reaction, speculating that RAMYG8b contains at least the 3 'end portion of the RAMYC26 gene. Therefore, RAMYG8c and RAMYG8b include the 5' end and 3 'end portions of the RAMYC26 gene. In addition to RAMYG8b and The total length of the three DNA fragments of RAMYG8c, RAMYG8a, RAMYG8d and RAMYG8e is about 5.6kb, so it is speculated that these three DNA fragments contain at least another ct-starch hydrolase gene, so RAMYG8 contains at least 2 ct-starch hydrolysis Gene group of enzyme gene E) NA pure strain. Table 1 shows that RAMYG6 and RAmy3D, RAMYG17 and RiUylA, RAMY G28 and RAmy3E are the same pure rice ct-starch hydrolase gene group DNA pure strain, P0S137 (RAMYC6) is a pure cDNA strain derived from RAMYG6, P0S103 (RAMYC2 7. ) Is a pure cDHA strain derived from RAMYG17, and ct-Amy3 (RAMYC26) is a pure cDNA strain derived from RAMYG8. RAmylA (RAMYG17) has a higher DNA transcriptional activity than other known α-starch hydrolase genes in rice during the piercing stage and root and leaf tissues of rice. (Huang et al., 1990a) ◦RAiny3 (RAMYG28) performed the most strongly in immature rice seeds (Huang et al., 1990a) and when the medium was subjected to sugar deficiency induction (Kuo, 1990). RAmy3D (RAMYG6) is the most expressed in rice callus (cal lus) (Huang etal., 1990a) ° The similarities between RAMYG6 (RAmy3D) and RAMYG28 (RAmy3E) include: (a) both on the chromosome The transcription direction is the same, (b) all have 2M inserts and the length of the inserts are similar, (c,) unmodified protein (Pr · epr · 〇tei η) the number of amino acids is similar, RAmy3D is 435 The amino acid, RAmy3E is 437 keto acid, (d) can be expressed in a large amount in callus (Huangeta 1., 1990a). Although RAMYG6 and RAMYG28 have these similarities and are in the same position, please read first. Please read the notes on the back Λ S-¾ fill in the package f. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 45 Ministry of Economic Affairs A7 _____B7_ printed on the chromosome of the invention by the Consumer Cooperative of the Central Bureau of Standards V. Invention description (Mojin), but the results of the cross-hybridization by DNA using the slot-blot method belong to different ethnic groups. Ethnic group 5 (Huang et al., 1990b). Comparison of the amino acid sequence of the signal wins chain ’RAMYG28 is more similar to wheat ’s Amy3 (Table 2), and RAMYG6 is significantly different from other cereal α-starch hydrolase genes. The protein secreted by plants out of cells can be divided into two categories according to their structure and function. The first type is proteins that do not have enzyme activity. The hydroxyproline of the oligosaccharide and the peptide bond are bound together via a 0-glycosidic bond to form a non-enzymatic secreted protein (Fincher et al., 1983). The second type is protein with enzyme activity. The oligosaccharide and Asn-X-Thr / Ser (X represents other amino acids except i, Asp or pro) in the peptide bond sequence are bound by N-glycosidic bond Together to form a secreted protein with enzyme activity (Akazawa et al., '1985). 3 The main difference between the α-starch hydrolase of rice seeds and the α-starch hydrolase of other cereal seeds is that there are many mannose and The compound oligo- will bind to the mature peptide bond of rice (Mitsui & Akawaza, 1986), but the mature α-starch hydrolase of barley and wheat has not been glycosylated. In this case, RAMYG17 has a hypothetical N-glycosylation position at positions 2450 and 3380 (Figure 8), and the amino acid sequences are Asn-Leu-thr and Asn-gly-Thr, respectively. Asn-Leu-Thr is located in the signal peptide chain, when the protein is secreted into the ER lumen, the signal-like bond will be cut off, so the mature α-starch hydrolase produced by RAMYG17 is predicted to be only at A s η- g 1 y-T hr is converted into a protein by N-glycosylation. (Please read the precautions on the back and fill in this page first)

, -ST line This paper scale is applicable to China National Standard (CNS) A4 specification (210x 297 mm) 321685 A7 ______B7_ V. Description of invention (required)

Kumasai et al. (199〇) used p〇sl〇3 (RAMYC27) to colonize yeast cells' to collect the secreted α-starch hydrolase in the medium, if the deglycosylated drug TFMS (trifluoro Methanesulfonic acid) treated ct-starch hydrolase 'result Alpha-starch hydrolase treated with TFMS has a molecular weight lower than that of alpha-starch hydrolase without TFMS treatment, proving that mature alpha-starch hydrolase produced by p〇si03 Indeed after glycosylation. RAMYG6 only has a hypothetical N-glycosylation site at the signal winning bond Asn-Thr-Ser (Figure 12), while RAMYG28 predicts that no place can be N-glycosylated (Figure 11). If the N-glycosylation site follows the Asn-X-Thr / Ser principle, the mature, ex-starch hydrolase produced by RAMYG6 and RAMYG28 will not be glycosylated (0 'Ne Π I et a 1. , 1.9 post). This is different from Miyata et al. (1982) 's conclusion that all rice α-starch hydrolases have N-glycosylation. Table 2 lists the comparison of the amino acid sequences of the rice, barley and wheat signals. RAMYG17 has 2 hypothetical translation starting points. The translation starting point starts from the first methionine, and its signal winning chain has a length of 31 amino acids. When thiamine starts, the signal wins key contains 25 amino acids. Von Heijne (1985) once divided the signal winning chain into three value regions of η, c and h. The η region is located at the N-terminus of the signal winning peptide chain, and consists of small peptides with one or two positive electric valences. The c region is located at the C-terminus of the signal peptide, consisting of approximately 5 amino acids. The h region is located between the η and c regions, and is composed of about 7 amino acids to form a hydrophobic core of the signal peptide chain. 1.—Table 2 shows that barley and wheat are almost in the c region of the signal peptide chain. The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm). Please read the notes on the back • Install-爿Fill in this page) Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy 47 ^^ 1685 A7 B7 V. Description of Invention (Mo Γ) Conserved amino acid sequence (SLASG), therefore, it is speculated that this sequence may be a signal peptide The position of the chain identification (Kumagai et al., 1990). However, the signal peptide in rice _ this region of c has no such conservative amino acid sequence. In the η region of the peptide bond of the ot_dian powder hydrolase signal in Table 2, except that RAMYG28 has three positive electricity prices, the rest have one or two positive electricity prices. In the h region of all signal peptide chains, there is a small hydrophobic core composed of six or seven consecutive hydrophobic amino acids. This hydrophobic peptide may serve to bind to the hydrophobic bilayer lipid of the endoplasmic reticulum membrane, thereby guiding the polypeptide into the lumen of the endoplasmic reticulum. In addition, the signals of α-starch hydrolase listed in Table 2 do not have acidic amino acids, which is worth noting. The Sanya rice α-starch hydrolase promoters obtained by the Employees ’Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs printed this issue have been constructed as vectors to control GUS performance (Figures 14, 15, 16, 17). Among them, RAMY G6- Two GUS expression vectors such as 132 and RAMYG23-132 (I) form a termination translation signal TGA at the position of multiple implantation sites where the DNA sequence of the peptide chain of the starch hydrolase and the GUS gene are connected (Figure 15D , 16D), therefore, the N-terminus of the GUS protein precursor tt translated by these two bow GUS expression vectors should not contain the signal of α-starch hydrolase, so the GUS protein cannot be secreted out of the cell. Using the method of electric penetration to transfer GUS expression vector into rice suspension culture cells, it has been determined that pHE 132, pAE 132 and RAMYG6-132 can make the GUS gene express (Figure 18), so it is proved that the PHE132 constructed in this experiment. , PAE 132 and RAMYG6-132 and other promoters-GUS gene fusion vector in water _ 'rice cells can indeed perform normally' and predicted ΡΗΕ 1 3 2 and p AE 1 3 2 This paper scale is applicable to the Chinese national standard (CMS ) A4 specification (210X297 mm) -48-A7 ___ _B7 5. Description of invention (slave) The G US protein produced will be secreted out of the cell. Based on the discovery that the lack of sulphur source in the medium earlier by the inventors of this case can induce the expression of the α-starch hydrolase gene and the above experimental results, it can be concluded that the promoter and signal peptide chain of the α-starch hydrolase gene are used to guide protein secretion Persistent, can be engaged in the study of gene regulation and protein secretion mechanism, and then replace the GUS gene with other desired human genes, under the control of the promoter of α-amylase gene, suspended in plants (such as rice) Cultured cells are treated with sulphur-deficient treatments, especially sugar-deficient treatments, to engage in mass production of engineered proteins for the benefit of the population. Although the present invention is described by the above preferred embodiment, but as can be understood by those skilled in the art, through the above teachings, this case can still produce various changes. The patent protection to be claimed shall be limited by the definition of the attached patent application scope and its equivalent replacements. I ---; ----------- IT ------ (please read the precautions on the back h &quot; fill in this page) (printed copy of Beigong Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X 297mm) A7 _B7 V. Description of the invention ($?) Statement of matters: 1. About the C (-starch hydrolase gene pure strain mentioned in the description of the invention of the case ( The name of clone): RAMYG-- 〃pure strain referred to in the description of the invention of this case, where the letter G in the letter represents the pure strain is `` genome DNA pure strain &quot;, and it is called `` RAMYC-- 〃Pure strain, where the C letter pseudo represents the pure strain as * cDNA group pure strain〃.-2. The definition in the independent claims of items 1, 10 and 26 of the scope of the patent application of this case includes "α-starch" The a Amy 6, ot Aray 7, ot Ainy 8 or ct AmylO gene in the promoter region of the hydrolase gene, which corresponds to the 12-value pure strain obtained by the inventor of the present invention in the present invention (please refer to the 44th specification of the present case) The description at the bottom of the page to the top of page 45). The inventor of the present case adopted the name of his laboratory for the original pseudo of the obtained pure strain. No. to mark, and then to publish a paper, rename these pure strains to the international naming method and rename them, where: ot iUy6 corresponds to RAMYG26 described in the specification of this case; a Amy7 corresponds to the description in this specification The described RAMYG17; cx Amy8 corresponds to the RAMYG28 described in the specification of this case; a AmyiO corresponds to the RAMYC30 described in the specification of this case, and a Amy30] corresponds to the RAMYG6 described in the specification of this case. 3. In this case The results shown in Figures 18B and 18C are the performance results of 'PHE132 &quot; transfer into rice suspension culture cells, and the transfer of the other two GUS expression vectors PAE132 and RAMYG6-132, because The results are assumed to be the same, so no photos will be presented. The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X 297mm) 32 ^ 685 A7 B7 V. Invention description (political) Table 1: Rice α-starch The nomenclature of the hydrolase gene and its performance in the cultured rice cells. The table lists the rice ct-starch hydrolase bases individually owned by this laboratory and the laboratory of R 〇drig U e ζ in the United States DNA and cDNA group of pure strains of pure strains. Table understood by those with RAMYG6 RAmy3D, RAMYG17 and RAmylA, RAMYG28 the same OT _ RAmy3E temple powder hydrolase gene cluster DNA pure strains. And RAMYC6 and mouth 05137, 1? 0 ¥ 027 and? 05103, [^ «¥ 〇26 and" -3 11 ^ 3 are the same pure cc-starch hydrolase cDNA strains. The pure ct-starch hydrolase cDNA strains such as RAMYC6, RAMYC26, RAMYC27, and .RAMYC28 are derived from pure DNA strains of a-starch hydrolase gene groups such as RAMYG6, RAMYG8, RAMYG17, and RAMYG28, respectively. RAmy3D (RAMYG6) showed the strongest performance in rice calli 'and the sugar-deficient treatment in the culture medium of rice suspension culture cells' RAmy3E (RAMY G28) performed the strongest. Table I

Rodriguez fs Lab * Calligraphic performance in the class Level b Sucrose-deficiency induced z Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

Til's Lab Genome cDNA Genome RAMYG6 RAMYC6X b RAmy3D RAMYG5 (RAMYG26) RAMYC267 RAMYGI7 RAMYC277 RAmvlAc (RAMYG27) (X0Sg2) RAMYG25 RAMYC26y RAmy3Eb RAMYC3〇y cDNA pOS137 ot-Amyd , Davis, &lt; bHuangetal &quot; 199〇a cHuang etal., 1990 !, d〇Neill etal., 199〇 • GenBank Data XHwa, 1991 * rT2〇u, 199〇E Kuo, 1990 (?)

95616, USA (?) I —Line setting--Η i "(Please read the notice on the back side first and fill in this page) This paper size is applicable to the Chinese National Standard (CNS> A4 specification (210X 297mm) 51 A 7 B7 5. Description of the invention (f) Table 2: Comparison of the amino acid sequence of the peptide peptide bond of the cc-starch hydrolase of gramineous plants. Conserved sequence in the C-terminal region of the acid sequence. However, there is no such conserved sequence in the rice signal bond. The thick horizontal line indicates the hydrophobicity composed of about 6 to 7 amino acids in the signal peptide. The core of the signal. In the N-terminal area of the signal win key, except RAMYG28 has three positive electricity prices, the rest have one or two positive electricity prices. Table 2 (please read the precautions on the back before filling this page) · Printed by the Employees Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs

Species isomers Pure strain amino acid sequence Rice Unknown RAMYG17 ++ MQVLNTMVNKHFLSLSVL IVLLGLSSNLTAG Rice Unknown RAMVG6 MKNTSSLCLLXLVVLCSLTCNSGGA Rice Unknown RAnRG28 Shishichuang MGKHHVTLCCVVFAVLCLASS LAQA Wheat Unknown Amy3 nGKHSATLCGLLVVVL Wheat High Class LAGGPI-LAGGLA4 ρΠ / 匚 ++ nANKHLSLSLFLVLLGLGLLASLASG Barley high pi Amy64 nANKHLSLSL.FLVLLGLSASLASG Barley high Pi Amy46 Ten ten MANKHMSKSLFIV.LLGLSCSLASG Barley high Pl 1-28 Ten ten MANKHLSLSL.FLVLLGLSAGLASG Wheat high Pi Amy I MANK FLVLLGLSASLASG Barley low pl CloneE nGKNGSLCCFSLLLLLLLAGLLASG Barley low pl Amy32 / B nGKNGNLCCFSLLLLLL AGL ASG Wheat low p Amy2 / 54 hGKNGNLCCFSLLLLLL GF ASG Threading This paper size is applicable to Chinese national standards (CNS) A4 specifications (210 &gt; Description (α) Α7Β7 MS medium nh4no3 KN〇3 stock solution 62.5 g / 1L 95g / 1L (5〇X) MS medium ': No. 1 ——5: No. 6 ΎΡΝ. Muscle: (1000 ml) 2 0ml Ξ Cacl2-H20 22 g / 1L (5〇X) 10ml (lOg / iL) (10 uM) a MgS04 * 7H20 MnS04H20 ZnS04 * 7H-0 15.5g / 1L 〇.545g / 1L 0.425 g / 1L (5〇 X) 2.4 -D: 10 ml sucrose-: 3〇g adjustment pHξ: 5.δ Θ kh2po4

_ 6,5g / lL H3BO3 0.31 g / 1L KI 41.5 mg / 1L Na2M〇〇4 · 2H2〇 12.5 mg / 1L (5〇X)

0 Na2-EDTA Fe2S04- 7H20 14.65 g / 1L 1.39 mg / 1L (5〇X) ΕΓ CuS04'5H20 'c〇a2- 6h2o 25 mg / 1L 2¾ mg / 1L (1000X) [7] T-PJi-: Vitamin BrHCl ig / 1L Vitamin B6 -HC1 lg / 1L (100 X) Niacin log / 1L Fu Ruiyi (please read the notes on the back to fill in this page)-Pack. Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economics The size of the paper used is in accordance with the Chinese National Standard (CNS) A4 (210X297 mm). 53 Printed by the Ministry of Economic Affairs, Central Standards Bureau, Employee Consumer Cooperative. B. V. Description of Invention (r /) Washing Medium ”CPW7.4! 1. CPW A: X / 1000 ml KH2P〇4 1-36 g 1 KN〇3 〇-5〇5g 1 MgS04 · 7H2〇12.3 g; KI ΟΟΟδδ CuS04-5H2〇0.00125 g plus H20 to 1000 ml 2. CPW B: 5 〇X / l〇〇〇ml

Cad2-2H20 74 g plus -H20 to 1000 ml 3- CPW 7.4 CPW A: 2 cc CPWB: 2 cc mannitol · 7.29g plus H20 to 100ml and adjust—PH to Fu Rui II 54 Standard (CNS) Α4 specification (2 丨 0Χ 297mm) V. Description of invention (Ο) A7 B7

Plate culture medium KPR ΚΠ 50 X / 100 ml NH4N〇3 3g kno3 9.5 9 MgS04- 7H20 1.5 g KH2PO4 0.05 g Kcl 1.5 9 6. Add H20 to 彳 00 ml 2. B5: 50 X / 100 ml Νθ2Μ〇04 · H2 0 12.5 mg KI 3.75 mg h3bo3 15 mg MnSQ4-H2〇50 mg 2nS04- 7H20 10 mg CuS04-5H20 0.125 mg l C〇Cl2- 6H20.10.1 mg; add .h2〇 to 100 ml 3. MS2 5- TPN: 100 X / 100ml Niacin 10 mg Vitamin B6 10 mg Vitamin · 100mg with H2〇 to 100 ml KPR (IX): KM B5 MS2 MS5 TPN Inositol 2.4-D NAA kinetin glucose sucrose 2 ml 2 ml 2.8 m ] 2 ml 1 ml 1ml (Ig / 100ml) 1 ml (22mg / 100ml) 1 ml (1S.6mg / 100ml) 0.25 ml (21.5mg / i〇〇mi) 10 g 0.025 g

Cac) 2- 2H20 plus 50 X / 100 ml 2.2 g add H20 to 100 ml and adjust the pH to 5.8 H2〇 to 100 ml 4. MS5 50 X / 100 ml ^ 0.139 (A 1.Θ63 under Ne2ED! Add H20 to 100 ml Three copies of the paper printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs are printed in accordance with the Chinese National Standard (CNS) A4 specifications (210X29? mm) 55 The A7 Β7 printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs Guang &gt;) Round-shaped illustration: Figure 1: Using the 1? / \ &Gt; ^ 030 coding region as a probe to screen a pure strain of the 14 bowel gene group, further analyze whether it is an α-starch hydrolase gene. A) (C): The pure strain of genomic DNA screened from the rice genomic library was cut with the restriction enzyme Sa II autophagic phage DNA, separated by agar colloid electrophoresis and stained with ethidium bromide. Photos, where RAMYG19 and RAMYG17 may be the same pure strains. (B) (D) Transfer the DNA from the agar colloids in (A) and (C) to the gene sieve membrane by Southern blot method. The probe hybridization of the RAMYC30 coding region revealed 4a (9kb), 4b (2.3kb), 5a (6.5 kb), 5b (3.2k b), 5c (2.3kb), 8a (4.8kb), 8b (3 kb), 8c (1.2kb), 8d (0.5kb), 8e (0.35kb), 15a (4.5kb), 17a (4.3kb) , 21a (7.0kb), 22a (4.5kb), 22b (3.0kb), 23a (3.8kb), 23b (2.2kb) all showed hybridization reactions, so it is inferred that these DNA fragments may contain 〇t-starch hydrolase gene. Several of his pure strains, such as RAMYG10, RAMYG11, RAMYG 14, RAMYG18 and RAMYG20, have no positive reaction, so they may not contain the ct-starch hydrolase gene, or the gene homology of the starch hydrolase gene and probe DNA Very low. Figure 1 (A) and (C) the DNA fragments and probes are reacted, all identified by circles, "M" represents the DNA marker of the Ikb ladder. Figure 2 ·· Determine the α-starch hydrolase gene The 5 'end part: &lt; _ (A) (C) (Ε) (G): Use Sal ί to adapt the rice genome DNA autophages to the original paper wave scale to the Chinese National Standard (CNS) A4 specification (210X297 mm)- ---------- Install-(please read the notes on the back t fill in this page), 11 lines · ----- 82 ^ 685 Printed A7 B7 by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Fifth, the description of the invention (ίζο DN Α separation. Electrophoresis pictures after separation by agar colloid electrophoresis and staining with ethidium bromide, among which RAMYG18 and RAMYG28 may be the same pure strain. (B) (D) (F) (H): Using the three probes made at the 5 'end of the coding region of RAMYG28 and RAMYG30 and the 3' end (HK350) of HS 501, through the results of molecular hybridization, RAMYG6, RAMYG8, RAMYG 17, RAMYG18, RAMYG21, and RAMYG28 all show positive hybridization reactions, so these five strains of pure strains are likely to contain the promoter site of the α-starch hydrolase gene. The coding regions of RAMYG18 and RAMYG30 in Figure 1 (D) do not show a positive hybridization reaction, but here show a positive hybridization reaction with the 5 'end of RAMYC28. Figure 3 The selected α-starch hydrolase gene group DNA and the four α-starch hydrolase cDNA (RAMYC26, RAMYC27, RAMYC28, RAMY C30) 3 'end of the fixed probe caused by the hybridization reaction, to . To confirm whether these cDN A may be derived from the DNA of the isolated gene group ° (A) (C) (E): After the pure plant of rice gene group DNA was cut through the restriction enzyme Sal I autophagophyte DNA, DNA electrophoresis photos after separation by agar colloid electrophoresis and staining with ethidium bromide. The DNA fragments and the probes in the figure have responders, which are marked by circles. (B) (D) (F) (G): The X-ray film after the hybridization of D N A in Figure (A) (C) (E) with the holding gene probes of four c D N A to-be-determined genes. Figure 4: (A) (B) RA Μ YG 1 7 a (4.3 kb) and RA Μ YC 3 0 coding area 5, the original paper size is applicable to China National Standards (CNS) A4 specifications (210X: ! 97mm) 57 --------- 7 outfit-(please read the notes on the back first and fill in this page) A7 B7 printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economy V. Description of invention (ο There is a hybridization reaction, and the double digestion of two restriction enzymes, Sal I and EcoRI, can reduce DNA from 4.3 kb to 1.2 kb and still have a hybridization reaction, thereby facilitating DNA sequence analysis. (C) (D ) RAMYG18a (7.5kb) has a positive hybridization reaction with the 5 'end of the RAMYC23 coding region. Using the double digestion of Sa 1 I and EcoRV restriction enzymes, the DNA can be further reduced from 7.5 kb to 1.8 kb while still having impurities • Orthogonal reaction to facilitate DNA sequence analysis. Figure 5 The results of the hybridization reactions in Figures 1, 2, and 3 are briefly summarized as shown. The seven probes used in the experiment are all different The size of the rectangular pattern represents the strength of the positive and negative reactions of the hybridization. The larger the case, the positive and negative of the hybridization. The weaker. The dotted line in the figure indicates that the same DNA fragment has a positive hybridization reaction with the two or three probes in the dotted line. Figure 6: In Figures 1 and 2, part of the coding with the α-starch hydrolase gene The probe at the 5 'end is a hybridized DNA fragment, which is cleaved with the restriction enzyme Sal I autophages DNA, separated by electrophoresis on agar colloid, and then separated from the colloid [for details, see materials and methods ( 1.2.3.1)], and then insert to limit the cut plastid PB 1 uescr i Pt. The size of these DNA fragments and the position of insertion in pBluescript are shown in (A) to (Μ). Figure 7: 'RAMYG17a t and λ 0Sg2 (Huang et a 1 ·, 1 990b.) DNA sequence paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 58 --------- installed-: -((Please read the precautions on the back before filling in this page) A7 ______B7 printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economics. Comparison of the invention description (/ 4). The DNA lengths of RAMYG17a and λ 0Sg2 are 4.3 kb, but the 5 'end of the elder brother 1731 is lkb longer than λ 0Sg2, and at the 3' end is shorter than λ 0Sg2 lkb DNA fragment (about 3.3 kb) within the dotted line represents the DNA sequence identical RAMYG17a 〇Sg2 of λ. Figure 8: DNA sequence analysis of published person 〇3 叾 2 (1 ^ ”(: 1 go) (1 ^ 3“ 61: 3 1., 199013) braces 1? $ At (2100 ~ 3250) means RAMYG17a2 , Is a DNA fragment that has been determined by the inventors of the DNA sequence. The double line indicates the TATA box, the right arrow indicates the transcription initiation sequence, and the curve indicates the α-starch hydrolase gene of rice, barley, wheat 5 Conserved sequences are contained in the 'terminal part. The pyrimidine box at the coffin line is a conserved sequence contained in the 5' terminal part of many genes induced by GA. The single line indicates the amino acid sequence of the signal peptide chain, The upward arrow is the position of the DNA sequence cut by EcoN I. And (1590) and (770) are the positions of Hind M and AccI. The dotted line indicates the amino acid sequence position that may bind to calcium ions, the thick line Mark the location of possible N-glycosylation. Figure 9: The 5 'end of RAMYG 17 contains a promoter that can be cut to different lengths using restriction enzymes. The total length of RAMYG17 is about 12.6 kb, and the black solid area is RAMYG17a2. Is to complete the DNA sequence analysis and found that the DHA sequence with λ 〇Sg2 is completely The same DHA fragment. Using Sa 1 I can 'separate RAMYG17ai (4.3kb) from RAMYG17, RAMYG17ai -------- r—installed ------ ordered ------ line-- if (please read the notice on the back &quot; fill in this page first) The paper size is applicable to China National Standard (CNS) Λ4 specification (210X29 * 7mm) -59-59 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 7 B7 5. The restriction map of the invention (β) is shown in the figure. Using Hind Π, A cc I, Sa 1 I and EcoHI, RAMYG17ai can be cut into DNA fragments with different lengths and signal peptides. , Respectively called pHE (0.88kb), pAE (1.7kb), pSE (3.4kb). B, E, S, X, Xh in the figure represent the restriction enzymes BamHI, EcoRI, Sail, Xbal, Xhol. 10: The relative positions and lengths of RAMYG6 and RAMYG28 and the published RAmy3D and RAmy3E (Huang et al., 1990a). RAMYG28a and RAMY G6a are two adjacent α-starch hydrolase genes with 27bp in the middle. Transferred to the plastid. RAMYG6a and RAMYG6b may be connected to each other or there may be a gap, which is still to be known by restriction enzyme analysis. , R ^ YG6b and RAMYG6c are two connected DHA fragments. RAM'YG6b and RAMYG28az are the sites where the inventor of the present invention has determined the DNA sequence, and the length is 1.8 kb. In the figure, RAMYG28a, RAMYG28a2, RAMYG6a and RAMYG6c are all represented by the same sigmoid shape, which means that these DNA fragments and the probes made of the 5 'end of the RAMYC28 coding region all show a positive hybridization reaction. _ 11: DNA sequence analysis of rice α-starch hydrolase gene RAray3E (Huang et al., 1990) and RAMYG28aa DNA (790-1 1 10) in braces g $ represents the probe used in Figure 2 The position of the 5 end of the code area. The DNA sequence from above 1320 (left arrow) to -630 is the position of R / \ MYG28a2 (Figure 11), the minus sign indicates! ^ 1 ^ 02832 has more DNA sequences at the 5 'end than RAiny3E, with about 6 30 nucleotides. From the 3 'end below 1 5 9 0 (the arrow pointing to the right applies to the Chinese national standard (CNS) A4 specification (210X297 mm) 60 1 I nΛ— installed n I nn II -.- I line: (f ( Please read the precautions on the back-ff fill in this page) A7 B7 5. The description of the invention (ff)) is the same as the DNA sequence of RAMYG6a (please refer to Figure 10). The double line represents TATAbox, the curve represents rice, barley, wheat The 5 'end of the α-starch hydrolase gene contains similar sequences. The horizontal line indicates the possible amino acid sequence of the signal chain. There are 25 amino acids in total. The dotted line shows the amino groups that may bind to calcium ions Acid sequence part. Figure 12: DNA sequence analysis of rice α-starch hydrolase gene 〇my3E (Huang et al., 1990a) and RAMYG6b, RAM YG 6 c. Braces (ξ DNA at 1350 ~ 2130) The DMA sequence is the same as RAMYG6c, and the DNA sequence from the 5 'end above 1350 is exactly the same as RAMYG6b. I? AM: YG6b and RAMYG6C are connected by the recognition site of restriction enzyme Sail. The DNA between the two thick arrows (1380 ~ 1650) indicates the 5 'end of the RAMYC28 coding region used in the probe 2 Highly homologous DNA sequence. The double line represents the TATA box, and the curve is the conserved sequence contained in the V-terminal part of the α-starch hydrolase gene of rice, barley, and wheat. The single line indicates the possible signal of the amine group of the peptide chain Acid sequence, there are 2 amino acids in total. The dotted line indicates the possible calcium ion binding amino acid sequence position. Group Wei represents the possible glycosylation site. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 13: Transplantation of GUS gene onto plastids with alternative marker-hygromycin resistance gene (A) Map of plastids pBSI, pBXE, pBSI. At the 5 'end of GUS gene, there are more PBXE than pBSI A bitter acid, and pBS II also has one more nucleotide than PBX ϋ, thus causing its reading structure ...- Each has a difference in the nucleotide, so that the promoter and part of the foreign gene 61 paper size is suitable for China National Standard (CNS) Α4 specification (210X297 mm) A7 _B7_ printed by the Consumer Labor Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs V. Invention description (ff) The gene can be properly combined with the GUS gene and still conform to the gene triple code (B) (C) (D) (E): Using Pvul to separate the GUS gene and the NOS terminator from pBSI, pBXI, and pBSM (Figure B), and the 5 'and 3' ends of this DNA fragment still carry There are multiple colonization sites of plastid p B 1 uescript. Insert this piece of DNA into pTRA132 (Figure C) cut with Hindi to obtain pBXH-132 and pBSDI-132 (Figure D). The translation direction of the GUS gene is the same as the translation direction of the anti-moisture gene. The translation direction of the GUS gene of pBSI-132 (Figure E) is opposite to that of the anti-moisture emblem gene. Figure 14: The site containing the promoter at the 5 'end of the q-starch hydrolase gene was transferred to a plastid containing the GUS and hygromycin resistance genes. (A) (B) (C) (D) (E): RAMYG17 was cut out with restriction enzymes. The promoter lengths pHE, pAE and pSE (Figure B), and then inserted into pBSI plastids cut with Smal (Figure A). As a result, three GUS expression vectors with no selectable symbols such as pHE-I (5.95 kb), pAE-I (6.85 kb) and pSE-I (8.55 kb) (Figure C) were obtained. Reuse the restriction enzyme PVU I to isolate the promoters of different lengths together with the GUS gene from pHE-I, pHE-I and pSE-I, and then re-transplanted to the restriction enzyme Hindi cut plastid PTRA132 (Figure D ), The results obtained three GUS expression vectors with anti-moisture emblem gene (Figure E), of which PAE132 and PSE132 have the same GUS gene translation direction and anti-moisture gene translation direction, while PHE132 GUS gene translation direction is Contrary to the anti-moisture emblem-gene translation direction. (Please read the precautions on the back first and then fill out this page) • The size of the paper for binding and binding is applicable to the Chinese National Standard (CNS) A4 specification (2 丨 Ο X 297mm) -62- V. Description of the invention (60 A7 B7 Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (F): DNA sequence analysis at the junction of the three DNA segments pSE132, pAE132 and pHE132 with the 5 'end of the GUS gene. The first ATG is the α-starch hydrolase gene The starting point of the translation, the second ATG is the starting point of the translation of the GUS gene, the horizontal line _ indicates the DH Α sequence of the expected signal win chain. Figure 15: the ex-starch hydrolase gene 5 'end contains the start The position of the promoter is transferred to the plastid containing the G US and hygromycin resistance genes. (A) (B) (C) Using the restriction enzymes EcoNI and Sal I, the promoter portion of RAMYG6b (Figure 10) together with the signal wins. The DNA sequence of the strand is isolated from the plastid pBlue-scr ipt. Then the isolated promoter (Figure B) is inserted into the pBSJI-132 (圔 A) vector cleaved by the restriction enzyme Hindi to obtain RAM, / G6-132 ( 836kb) GUS expression vector (Figure C). The translation direction of GUS gene of RAMYG6-132 is the same. (D) ·: RAMYG6-132 and GU Analysis of DNA sequence at the 5 'end of the S gene. The horizontal line indicates the predicted DNA sequence of the signal. The nucleotide sequence TGA at the dotted line represents the stop code. Figure 16: α-starch hydrolase The 5 'end of the gene containing the promoter was transferred to the plastid containing the GUS and moisture resistance gene. Uj (B) (C): Using the restriction enzymes Apal and EcoRV, the promoter part of RAMYG28a2 (Figure 10) was combined with The DNA sequence of the signal winning bond was isolated from pBluescript plastid. Then the isolated promoter (Figure B) was inserted into pBS BI-132 (Figure A &gt; vector cut with restriction enzyme Hind SI) to obtain RAMYG28-132 ( I) (8.2 kb) GUS expression vector (Figure C). RAMYG28-132 (I) The translation direction of the GUS gene is the same as the translation direction of the anti-moisture emblem gene. Please-read first-read back * Note Matters ^ Who's Filling This page of the bound paper The size of the paper applies to the Chinese National Standard (CNS) A4 (210X29 * 7mm) 63 V. Description of the invention (6 /) A7 B7 (D): RAMYG23-132 (I) and GUS DNA sequence analysis at the 5 'end of the gene. The horizontal line indicates the predicted DNA sequence of the peptide bond. The nucleotide sequence TGA at the dotted line of the box represents the stop code. Figure 17: The promoter region at the V-terminus of the α-amylase gene is transferred to the plastids containing GUS and anti-hygromycin genes. U) (B) (C): Using the restriction enzymes Apal and EcoRV, the promoter part of RAMYG28A2 (Figure 10) and the DMA sequence of the signal peptide chain were separated from pBluescript. Then, the isolated promoter (圔 B) was inserted into pBSI-132 cleaved by the restriction enzyme CUI (Figure C). The translation direction of RAMYG28-132 (I) GUS cells is opposite to that of the hygromycin resistance gene. (D) i: RAMYG28-132 U) DNA sequence analysis at the 5 'end of the GUSGUS gene. The horizontal line represents the predicted DNA sequence of the signal peptide chain ---- ^ ----- ^ installed — I * I ((Please read the precautions on the back before filling out this page) Order the consumption of employees of the Central Bureau of Standards of the Ministry of Economic Affairs Printed by Cooperative Society Figure 18: Transplanting GUS expression vector into rice suspension culture cells by electro-penetration method, and performing GUS analysis after 3 days of culture. (A) Control cells use a promoter without ct-starch hydrolase gene , Only the vector with GUS gene was used for transfection, and it was reacted with X-glu after 3 days of culture, and no blue appeared. (B) The cells transformed with GUS expression vector were reacted with Xg lu after 3 days of culture. The blue ones in the figure indicate the cell pellets that the GUS gene can express. (C) The magnification of the blue fine roars after coloration by the &lt; -g 1 u reaction is suitable for China. Standard (CNS) Α4 specification (210Χ 297 mm) 64 3 Magic 685 Printed by A7 Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economics A7 ____B7_ V. Description of the Invention (έχ) Outline of the Diagram Β 日 Figure 1: Using RAMYC30 coding area 14 strains of pure strains selected by the probe, of which (A) and (C) are Photographs of DNA electrophoresis after separation by lipid colloid electrophoresis and staining with ethidium bromide, (B), (D) are the results of Southern blotting hybridization of DHA on agar colloids of (Α) and (C), and circle marks indicate DNA fragments Reacts with the probe, "M" indicates the DNA label of the lkb ladder. ^ Figure 2: Identify the 5 'end of the α-starch hydrolase gene: U) (C) (E) (G): Use Sal I to transfer rice Genome DNA is separated from phagocytic and somatic DNA, and separated by agar colloid electrophoresis and electrophoresis after ethidium bromide-color electrophoresis; (B) (D) (F) (H): using RAMYG28 and RAMYG30 encoding Molecular hybridization results of the three probes made at the 5 'end of the region and at the 3' end (HK350) of HS 501.圔 3: The hybridization results of the selected α-starch hydrolase gene group DNA and specific gene probes made at the 3 'end of four α-starch hydrolase cDNAs (RAMYC26, RAMYC27', RAMYC28, RAMY C30), of which U Γ (C) (E): The pure DNA of rice genomic DNA is cut by restriction enzyme Sail autophages DNA, separated by agar colloid electrophoresis and stained with ethidium bromide. The circle mark indicates DNA fragments Reacts with probes;, (B) (D) (F) (G): 圔 (A) (C) (E) DNA after hybridization with four cDHA holding gene probe hybridization X-ray film. Figure 1-(Please read the precautions on the back to fill out this page) Binding-Binding-Line-This paper is again applicable to China National Standards (CNS) Α4 specifications (210X297 mm) A7 B7 Ministry of Economic Affairs Standards The bureau employee's consumer cooperation du printed five, invention description α winter) U), (B) shows that RAMYG17a (4.3kb) and RAMYC30 coding region 5 'end hybridization positive reaction, using Sal I and EcoRI two restriction enzymes Double digestion; (C), (D) shows the hybridization of double digestion of the two restriction enzymes Sal I and EcoRV at the 5 'end of the coding regions of RAMYG18a (7.5kb) and RAMYC28. Fig. 5: &quot; 'Simple summary of the results of the hybridization reaction from Figures 1, 2, and 3, where the seven probes used in the experiment are represented by different rectangular patterns, and the size of the rectangular pattern represents the hybridization The Qiangqin of the positive reaction, the dotted line in the middle of the box, indicates that the same DMA fragment and the q or three probes in the dotted line have hybridized positive reactions. Fig. 6:-The part shown in Fig. 1 and Fig. 2 and the coding of α-starch hydrolase gene. The probe at the 5 'end> was a hybridization positive reaction DNA fragment, which was inserted to cut with restriction enzyme Sail The plastid of pBluescript, and the size of these DNA fragments and the position j inserted in pBluescript are shown in (A) to (M). Fig. 7: Comparison of the DNA sequences of RAMYG17ai and A0Sg2 (Huang et al., 1990b), where the DNA fragment (approximately 3.3 kb) in the dotted line indicates that the DNA sequences of RAMYG17a and λ OSg2 are identical. Figure 8: 'Published A0sg2 (RAMYClA) (Huangetal ·, 1990b &gt; (please read the notes on the back-S to fill in this amaranth). Binding. The paper size of the binding book is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) A7 _____B7__ printed by the Employees ’Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy V. DNA sequence analysis of the invention description (6?), In which the braces (J $ place (2100 ~ 3250) indicate RAM YG17a2, and the double line indicates TATA box , The right arrow indicates the transcription initiation sequence, and the curve indicates the conserved sequence contained in the 5 ′ end of the α-starch hydrolase gene of rice, barley, and wheat. The frame line is the pyrimidine box, and the single line indicates the signal of the peptide chain. Amino acid sequence, the position of the DNA sequence cut by EcoNI is at the upward arrow, while (1590) and (770) ^ have the role of Hind H [and AccI ', the dotted line indicates the amino group that may bind to calcium ion The acid sequence position, the thick line indicates the possible N-glycosylation position Ο Figure 9 :, MMYG 17 5 'end containing the promoter site can be cut out with restriction enzymes of different lengths (total length is about 12. 6kb), Where the black solid area For RAMYG17a2, the restriction map of RAMYG17ai is shown in the figure, while v Hindi, AccI, Sail, and EcoNI refer to RAMYG17adJ〇 as fragments called PHE (0.38kb), PAE (1.7kb), PSE (3.4kb), B, E , S, X, Xh represent the restriction enzymes BamH I, EcoRI, Sail 'Xbal, X h ο I ° Figure 10: The relative position and length of RAMYG6 and RAMYG28 and the published RAmy3D, RAmy3E (Huang et al., 1990a) , Where RAMYG28a and RAMYG6a are two adjacent α-starch hydrolase genes, and RAMY G6b and RAMYG6c are two DNA fragments that are connected; in the figure, RAMY • G28a, RAMYG28a2, RAMYG6a and RAMYG6c are all in the same pattern (please first Read the precautions on the back and then fill out this page) -The size of the binding and binding paper paper is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) -67-V. Description of the invention (&lt; Γ) Figure 11 Figure 12 A7 B7 DNA sequence analysis of the rice alpha-starch hydrolase gene RAmy3E (Huang et al., 1990) and RAMYG28aa printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, in which DNA (790-11 10) in braces g $ indicates Figure 2 of the coding region of the probe RAMYC28 used in Figure 2 The position of the DNA sequence from above 1320 (left arrow) to -630 is listed as the position of RAMYG28a2. The minus sign indicates that the 5 'end of RAMY 02832 has more DNA sequences than RAmy3E, from the 3' end below 1590 (to Right arrow) is the same as the DNA sequence of R AM YG 6 a, the double line indicates the TATA box, the curve represents the similar sequence contained in the 5 ′ end of the α-starch hydrolase gene of rice, barley, and wheat, and the horizontal line indicates The amino acid sequence of the possible signal wins bond has a total of 25 amino acids, and the dotted line indicates the amino acid sequence part that may bind to calcium ions. f 'Rice ct-starch hydrolase gene R Amy 3E (Huang et al., 1990a) and RAMYG6b, RAMYG6c DNA sequence analysis, in which the DNA within the large house number ξ $ (1350 ~ 2130) represents its DNA sequence and RAMYG6c The DNA sequence from the 5 &gt; end above 1350 is exactly the same as RAMYG6b. RAMYG6b and RAMYG6C are connected by the recognition site of restriction enzyme Sa Π. The DNA between the thick arrows (1380 ~ 1650) represents the probes used in FIG. 2. The 5 ′ end of the coding region of the RAM YC2 8 has a highly homologous DNA sequence. The double line indicates the TATA box, and the curve is the α-starch hydrolase gene of rice, barley, and wheat. Please-read first-% 背-έ 之Matters needing attention The paper size of the binding line is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 68 A7 _B7 _ V. Description of invention) Conserved sequences are contained at the 5 'end of the single line, and a single line may indicate a possible signal peptide The amino acid sequence of the chain, the dotted line indicates the position of the possible calcium ion-bound amino acid sequence, and the thick line represents the site of possible glycosylation.圔 13: Transplantation of GUS gene into a plastid skeleton with an alternative symbol of a moisture-resistant emblem gene, where '(A): map of plastid PBSI, p BX Π, p BSH [; (B): Use Pvul to separate the GUS gene and NOS terminator from pBSI, pBX 1 and pBS I. (C): Insert this DNA segment into the plastid PTRA132 cut with Hind Π; (〇): get ρΒΧΙ -132 and pBSltt- 132 (Figure D), the translation direction of the GUS gene is the same as the translation direction of the anti-moisture gene. (E): The translation direction of the GUS gene of pBS I-132 is opposite to the translation direction of the anti-moisture emblem gene. Picture 14: Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs &amp;-the powdery hydrolase gene 5 'end containing the promoter is transferred to the plastid containing the GUS and anti-moisture emblem gene, where (A) ( B) (C) (D) (Ε): Using restriction enzymes, RAMYG17 was cut out of different promoter lengths ΡΗΕ, ρΑΕ, and pSΕ (Figure B), and then inserted into pBSI plastids cut with Smal (Picture / \). As a result, three GUS expression vectors, pHE-I (5.95kb), pAE-I (6.85kb), fepSE-I (8.55kb) (Fig. C), which were not available for selection, were obtained. Re-69 The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 3 ^ 1685 A7 B7 5. Invention description U ;?) 1 Use the restriction enzyme pvu I to combine promoters of different lengths with GUS The genes were isolated from pHE-I, pHE-I, and pSE-I, and then transferred to pTRA132 (Figure D), which was cut with the restriction enzyme Hind DI. Performance carrier (Figure E);

, (F): DNA sequence analysis where the three DNA segments of PSE132, PAE132 and PHE132 meet the 5 'end of the GUS gene. The first value ATG is the translation starting point of the ct- 'starch hydrolase gene * the second M ATG is the translation starting point of the G US gene, and the horizontal line represents the DNA sequence of the expected signal peptide chain. Figure 15: «Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. The C4 -starch hydrolase gene 5 'end containing the promoter is transferred to the plastids containing the GUS and hygromycin resistance genes, where (A) (B) (C) Using the restriction enzymes EcoNI and Sal I, the promoter part of RAMYG6b (Fig. 10) and the DNA sequence of the signal winning chain were separated from the plastid pBlu-escript. Then insert the isolated promoter (Figure B) into the pBSDI-132 (paint A) vector cleaved by the restriction enzyme Hindll # to the GUS expression vector of RAMYG6-132 (836kb) (Figure C); (D): RAMYG6- 132 DNA sequence analysis at the junction of the 5 'end of the GUS gene, the horizontal line represents the predicted DNA sequence of the signal peptide bond, and the nucleotide sequence TGA at the dotted line represents the stop code. Figure 16: The 5 'end of the α-starch hydrolase gene is transferred to the plastid containing G US and the anti-moisture emblem gene at the 5' end of the gene. Among which, the size of this paper is in accordance with the Chinese National Standard (CNS) A4 specifications ( 210Χ 297 mm) A7 B7 V. Description of the invention (MoP) (A) (B) (C): Using the restriction enzymes Apal and EcoRV, the promoter sequence of RAHYG28a2 (Figure 10) together with the DNA sequence of the signal peptide peptide from Plastid was isolated from pBluescript, and then the isolated promoter (Figure B) was inserted into the pBSM-132 (Figure A) vector cleaved by the restriction enzyme HindDI to obtain the GUS expression vector of RAMYG28-132 (I) (8.2kb) ( (Figure C); (D): DNA sequence analysis of RAMYG28-132 (I) at the 5 'end of the GUS gene, the horizontal line represents the predicted DNA sequence of the signal peptide chain, and the nucleotide sequence at the dotted line TGA stands for termination code. Figure 17 Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 18 The ct-starch hydrolase gene 5 'end containing the promoter is transferred to the plastids that eat GUS and anti-moisture emblem genes, of which (A) (B ) (C): Use the restriction enzymes Apal and EcoRV to separate the promoter part of RAMYG28A2 (Figure 10) and the DNA sequence of the signal peptide from pBluescript plastid, and then insert the isolated promoter (Figure B) to restrict PBSI-132 cleaved by enzyme Clal (Figure C); (D): DNA sequence analysis of RAMYG28-132 (I) at the 5 'end of the GUSGUS gene. The horizontal line indicates the predicted DNA sequence of the signal winning chain. The GUS expression vector was transformed into rice suspension culture cells by electro-penetration method, and the GUS analysis was performed after 3 days of culture. U) Control cells were transformed with a promoter containing no α-starch hydrolase gene, and a vector containing only the GUS gene. After 3 days of cultivation, X-glu 71 was used as the paper standard for China National Standards (CNS) A4 (210X297mm) A7 B7 Fifth, the description of the invention (6 $) reaction, and no blue appears. (B) Cells transfected with GUS expression vectors are colored with X-slu after 3 days of culture. The blue cells in the figure indicate cell pellets that can be expressed by the GUS gene. (C) Enlarged photo of blue cell pellets colored by Xg 1 u reaction ° (please read the precautions on the back before filling out this page). Packing and ordering 72 copies of paper printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs The scale is applicable to the Chinese National Standard (CNS) A4 specification (2 〖0X29? Mm) Printed by the Central Sample Bureau of the Ministry of Economic Affairs, the Consumer Labor Cooperative A 7 '------- B7 V. Description of invention W) References

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Reiss, B., Sprengel, R., Will, H. and Schaller, H. (1984). A new sensitive method for qualitative and quantitative assay of neomycin This paper scale is applicable to the national standard (CNS) A4 specification (2. .Produce) -77-(Please read the precautions on the back before filling in this page)-Install. A7 B7 printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economics 5. Description of invention (7〇phosphotransferase in crude cell extracts. Gene. 30: 211-218. Rogers, JC and Milliman.C. (1984). Coordinate increase in major transcripts from the high pi a-amylase multigene family in barley aleurone cells stimulated with gibberellic acid. J. Bio. Chem. 259 (19): 12234-12240. Rogers, JC (1985). Two barley α-amylase gene families are regulated differently in aleurone cells. J. Biol. Chem. 260: 3731-3738. Rothstein, SJ., Lahners, KN, Lazarus, Baulcombe, DC and Gatenby, AA. (1987). Synthesis and secretion of wheat a-amylase in Saccharomyces cerevisiae. Gene. 55: 353-356. Salmenkallio, M., Hannus, R., Teeri, TH and Kaupinen. (1990). Regula tion of a-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Reports. 9: 352-355. Sogaard, M. amd Svensson, B · (1990). Expression of cDNAs encoding barley a-amylase 1 and 2 in yeast and characteization of the secreted proteins. Qene. 94: 173-179. Tkachuk, R. and Kruger, JE (1974). Wheat a-amylase.II. Physical characterization. Cereal Chemistry.51: 508-529 . Tzou, WS (1990). Expression of α-amylase genes in rice aleurone layer. Tsing Hua University. Master Thesis. Von, HG (1984) * Analysis of the distribution of charged residues in the N-tenninal region of signal sequences: Implications for protein export in prokaryotic and eukaryotic cells. European Molecular Biology Organizational Journal 3: 2315-2318. Von, HG · (1985) Signal sequences. The limits of variation. J. Mol. Biol. 184: 99-105. Waldron, C., Murphy, EB, RobertsJ.L., Gustafson, GD, Armour, SL and Malcolm, SK (1985). Resistance to hygromycin B: a new marker for pl ant transformation studies. Plant Mol. Biol. 5: 103-108. Weickert, MJ. and Chambliss, GH (1990). Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis. Proc. Natl. --Acad. Sci . USA. 87: 6238-6244. (Please read the notes on the back before filling in this page)-Binding-The paper size of this book is applicable to the Chinese National Standard (CNS) A4 specification (21. *)-78-A7 ___B7 5. , Description of invention)

Yu, SM, Tai, YS, Goldman, S., Chuu, YJ., Ou-Lee, TM and Wu, R. (1990). Analysis of the promoter region of rice α-amylase genes. Structure and Function of Nucleic Acids and Proteins. 287-295.

Zhang, H.M., Yang.H · »Rech, E.L., Golds, TJ., Davis, A.S., Mulligan, B-J ·»

Cocking, E-C. And Darey, M.R. (1988) Transgenic rice plants produced by electroporation-mediated. Plant Cell Reports. 7: 379-384.

Zhang, W. And Wu, R. (1988). Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor Appl. Genet. 76: 835-840 .; outfit ·! *. i-^ (Please read the precautions on the back before filling in this page) ir Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs?

Claims (1)

32 Rbhomoto i ___1 D8 Printed and applied for patent scope No. 8 1 1 0 1 029 by the Zhenggong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. The date of amendment of the scope of application for the patent application was revised. June 1. 86. A method of preparing a vector with enhanced gene expression ability 'which includes integrating the promoter encoding the α-starch hydrolase gene derived from rice plants and the D ΝΑ sequence of the signal peptide chain into a carrier to regulate The expression of the structural gene encoding a desired protein in the vector, so that in the absence of sugar or sugar deficiency, the promoter of the α-starch hydrolase gene derived from rice plants can induce the encoding of the desired protein The expression of the gene of the rice plant, and the signal of the "-starch hydrolase gene derived from rice plants is better than N, can facilitate the secretion of the desired protein outside the host cell in order to recover the desired protein from the culture medium, wherein the alpha -The promoter region of the starch hydrolase gene is derived from rice aAmy6, a Amy 7 'a Amy8 aAmylO or aAmy3 gene 0 2. The method as claimed in item 1 of the patent application, which encodes the desired protein The induction of qualitative gene expression occurs under the condition of sugar deficiency in the medium. 3. A method as claimed in item 2 of the patent application, in which the induction of the expression of the gene encoding the desired protein occurs in the absence of sucrose, glucose or fructose in the medium. 4. The method as claimed in item 1 of the patent application, wherein the vector further contains a value marker gene, a reporter gene, an antibiotic-resistance gene, an enhancer or a regulatory sequence as a selection colony, so as to facilitate selection colonization Angiosperm host cells transfected by this vector. ‘5. The method as claimed in item 4 of the patent application, in which the vector Ye further contains a one-pump antibiotic-resistance gene as a selection marker. (Please read the precautions on the back before filling out this page) • The paper size of the binding and binding book applies to the Chinese National Standard (CNS) A4 (210X297 mm) 321685 A8 B8 C8 D8 Printed by the Employees Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 6. The scope of patent application 6. The method of item 5 of the patent application scope, wherein the antibiotics targeted by the antibiotic-resistance gene is kanamycin or hygr omyc i η ° 7. The method as claimed in item 4 of the patent application, in which the vector 傜 further contains a reporter gene as a selection record. 8. The method as claimed in item 7 of the patent application, wherein the reporter gene is a call-glucuronidase (/ 3-glucuronidase) gene. ^ 9. The method as claimed in item 1 of the patent application, wherein the desired protein is called _ glucuronidase. 1〇. A method for obtaining a desired protein by expressing a gene encoding a desired protein in the angiosperm cell, which includes: a) constructing a vector capable of expressing the gene in the angiosperm cell The vector includes a promoter region derived from the rice plant ct-starch hydrolase gene and a gene encoding a desired protein; b) transforming an appropriate angiosperm host cell with the characteristics of the vector; and c) formed by cultivation Trait-transforming host cells; d) introducing the cultivated trait-transforming host cells to a lack or sugar-free condition to promote the expression of the gene under the control of the promoter region; and e) recovering the expression of the gene Gene product, wherein the O (-starch hydrolase gene promoter region 傜 is derived from rice a A ray 6 'a Aray7, α Amy8, ot AraylO or ot Amy3 gene. 11. Such as the method of claim 10 , Where the promoter region is derived from a Aniy8 gene of rice. '12, such as the method of claim 9, 9 or 10, wherein the derivative is derived from J ----- j? — 装 — (please Read back first Please pay attention to this page and then fill out this page.) -The standard of the paper-based line is applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm) 2 321685 A8 B8 C8 D8 Printed by Patent application Fanyuan Ot-Dan powder hydrolase gene promoter region includes the promoter of α-source powder hydrolase gene and a DNA sequence encoding a peptide bond of α-starch hydrolase signal. The method of item 9, wherein the gene product is pseudo-recovered from the cell culture medium of the trait-transforming host cell. 14. The method of item 9 of the patent application scope, wherein the transfer of the vector to the host cell is pseudo-Agrobacterium Genus (Agrobacter i »m) -regulated trait transformation system, polyethylene glycol (PEG) -regulated trait transformation, poly-L ornithine method, calcium phosphate method, microinjection method, particle impact method, electricity Penetration method and ultrasonic method. 15. The method as claimed in item 14 of the patent application, wherein the transfer of the vector to the host cell is transformed by the soil rod®-regulated traits. 1 6. Upon application Patent scope The method of item 9, wherein the appropriate angiosperm host cell is rice, barley or wheat suspension culture cell. 17. The method of item 16 of the scope of the patent application, wherein the appropriate angiosperm host cell is a rice suspension culture cell. 18. The method as claimed in item 9 of the patent scope, wherein the lack of sugar or sugar-free condition is a condition of lack of sucrose, glucose or fructose. 19. The method as claimed in item 9 of the patent scope, wherein the gene product Yu is a protein of animal, plant or biological origin. 20. The method according to item 9 of the patent application scope, wherein the vector further contains a marker gene, a reporter gene, an antibiotic-resistance gene, an enhancer or a regulatory sequence as a colonization marker, which facilitates colonization by the vector The angiosperm host cells that have been transferred. L · (please read the precautions on the back before filling in this page). The paper size of the booklet is applicable to the Chinese National Standard (CNS) A4 specification (210X297mm) 3 ABCD 321685 6. Patent application scope 21. If the patent application scope The method of item 20, wherein the vector pseudo contains an antibiotic-resistance gene as a colonization marker. 22. The method as claimed in item 21 of the patent scope, wherein the antibiotic-resistance gene targets the antibiotic antibiotic kang Hui Su or hygromycin. 23. The method as claimed in item 20 of the Patent Scope, wherein the vector 傜 进 -step contains a value reporter gene as a selection marker. 2-4. The method as claimed in item 23 of the patent application, wherein the reporter gene is a Zhao- »'uronic acid oxonase gene. 25. The method of claim 9, wherein the desired protein is cold glucuronidase. 26. A method for obtaining a desired protein by expressing a gene i encoding a desired protein in angiosperm cells, which includes: a) constructing a vector capable of expressing genes in the angiosperm cells, the vector It includes a promoter J region derived from rice plant oc-starch hydrolase gene and a gene encoding a desired protein, the promoter region derived from α-starch hydrolase i includes the ct-starch hydrolase The promoter of the gene and a DNA sequence encoding the signal-bonding bond of the α-starch hydrolase; b) transforming an appropriate angiosperm host cell with the vector trait; c) transforming the trait resulting from cultivation in a cell culture medium Host cells; and d) introducing the cultivated traits into host cells to a lack of or sugar-free condition to promote the expression of the gene under the control of the promoter region; and-'e) recovering the gene from the culture medium The displayed gene products are in accordance with the National Standards (CNS) A4 specification (210X297mm) of this country. -4-ΙΓ ----- ^ I 装-(Please read the notes on the back first (Fill in this page again) Ordered by the Ministry of Economic Affairs, Central Standards Bureau, Negative Labor Cooperative Printing 321685 Printed by the Ministry of Economic Affairs, Central Standards Agency, Employee Consumer Cooperatives A8 B8 C8 D8, Patent Application Scope 1 where the Cf-amylase gene promoter region is derived A Amy6 Λ a Amy7 ^ a Amy8 ^ a AinylO ^ da Aray3 $ S 0 27 from rice • As in the method of claim 26, the promoter region 傜 is derived from the a Amy8 gene of rice. 28. The method of claim 26, wherein the transfer of the vector to the host cell is pseudo-regulated by the soil-genus-regulated trait transformation system, polyethylene glycol (PEG) -regulated trait transformation, Poly-L ornithine method, phosphoric acid punishment method, microinjection method, particle impact method, electro-penetration method, and ultrasonic method 〇 ~ 29 · The method as claimed in item 28 of the patent application, wherein the vector to the host cell The transfer is through the soil to maintain the genus-regulated traits to transform the system. 30. The method of claim 26, wherein the appropriate plant host cell is rice, barley or wheat suspension culture cell. 31. The method of claim 30, wherein the suitable plant host cell is a rice suspension culture cell. 32. The method of claim 26, wherein the sugar-deficient or sugar-free condition is a condition lacking sucrose, glucose, or fructose. 33. The method of claim 26, wherein the gene product is a protein of animal, plant or biological origin. 34. The method of claim 26, wherein the vector further contains a marker gene, a reporter gene, an antibiotic-resistance gene, an enhancer, or a regulatory sequence to serve as a colonization marker to facilitate colonization by the vector Transplanted angiosperm host cells. _ 35. For example, the method of applying for item 34 of the patent scope, in which the carrier further contains pseudo (please read the precautions on the back before filling this page) This paper wave scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297mm> 5 ® 2l685 A8 B8 C8 D8, and the patent application has an antibiotic-resistance gene as a marker. N \. 36. If the patent application scope is 35 The method of item 1, wherein the antibiotic-resistance gene is targeted to antibiotics such as konanaxin or hygroxin. 37. The method of item 34 of the patent application scope, wherein the vector further contains a value report The gene is used as a selection marker. 38. The method according to item 37 of the patent application scope, wherein the reported gene is a glucuronidase gene. 39. The method according to item 26 of the patent application scope, wherein The desired protein is called _glucuronidase. Please read first, -read; f on the back I, I, I, I, I, I //, poultry, T, I. Printed by the Ministry of Economic Affairs, printed by the Employee Consumer Cooperative I-I — ^ 7 /, I! -II -Is 11 I— j H This paper scale is applicable to the Chinese National Standard (CNS) Μ wash grid (210 X 297 mm) 6