TW402638B - 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 said 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 said 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 said GUS expression vector in said cells under the control of said 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

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, A7, B7, Description of Invention () Starch contains amylose and amylopectin, two types of "glucose polymer," which is the most important nutrient storage component of cereal seeds (AKazawa et ai. , 1985). In the early stage of grain seed penetration, aleurone cells synthesize α-amylase, α-amylase-glucosidase, and dextrinase and other enzymes secreted into endosperm to hydrolyze starch to glucose and maltose to provide embryo development Needed nutrients (Rogers, 1985). There is another enzyme involved in starch hydrolysis-starch hydrolase, which hydrolyzes starch into maltose and a small amount of ® glucose. In normal times, -amylases in the dried seeds bind to the protein bodies with disulfide bonds and become an inactive form in endosperm (Tronier et a 1 ·, 1970). The aleurone cells are also induced by G A 3 when the seeds are penetrated to produce proteases, which can destroy the disulfide bonds and release the active form of / 3-amylase. The above four enzymes are involved in the hydrolysis of starch during the process of seed hair penetration, but α- 丨 dian powder hydrolase produces the most and plays the most important role (Akazawa et al., 1988), of which GA3 is known It has a direct impact on the performance of α-alkaline hydrolase (Chandler et al 1987). When rice seeds are treated with GA 3, the newly synthesized α-amylase hydrolysate mRNA from aleurone cells will increase by 50 to 50. 100 times (O'Neill et al., 1990). In fact, the large expression of cc-amylase mRNA induced by GAa has provided an ideal research model for how plant hormones regulate gene expression (Ho et al., 1987). Rice, barley, and wheat 〇ί-amylase enzymes have been largely selected and further researched and analyzed. The results show that these cereal α-amylase isomerases are composed of a variety of α-amylase The size of the gene paper is applicable to the Chinese National Standard (CNS) Α4 specification (210X297 mm)-4 _ ^ ---- ,, ----- © packing__ (Please read the precautions on the back before filling this page} -Order A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Manufactured by (Invention) (Baulcombe et al., 1987; Huany et al., 1990; Knox, 1987). Barley and wheat seeds during germination Alpha-amylases secreted by aleurone cells are divided into two categories: higher and lower electrical points (Jacobsen, 1982; Lazarus et al., 1985). There are about 7 ot- Amylohydrolase genes belong to higher isoelectric point, and 3 to 4 gene belong to lower isoelectric point (Khursheed & Rogers, 1988). So far, there are 7 kinds of barley α-amylase cDNA and 9 kinds of α-amylase enzyme gene groups. DNA has been selected (Chandler et al., 1984; Deikman et al., 19 8 5; Khrusheed & Rogers, 1988; Knox et a 1., 1987; Whittier et a 1., 1987). The ct-amylase genes in wheat are divided into a-Amyl, a -Amy2 There are three groups of a -Amy3. Oc -Amyl has a higher mildew point, and cx -Amy2 is a lower isoelectric point. It contains at least 10 genes each and is expressed in germinated seeds (Col in et a 1. , 1985; A1 ison et al., 1988), ct-Amy3 contains 3 to 4 genes and is only expressed in immature seeds (Baulcombe et al., 1987). Studies on rice α-amylase enzyme genes However, like barley and wheat, the α-amylase enzyme gene could not be divided into higher electrical point groups and lower electrical point groups. In fact, Macgregor et al. (1988) used isoelectric point electrophoresis to find rice. The ct-amylase isomerases are concentrated at positions where the PI value is less than 5.5, so rice may not have an isoelectric enzyme with a higher isoelectric point. Huang et al. (1990b) used cross-hybridization experiments. The α- The amylolytic enzyme genes are divided into 5 groups and determined to be distributed on 5 chromosomes (Ranjhan et al., Manuscript in In preparation) 〇CTNeill et al. (1990) (Hong Please read the Notes on the back to fill out Page)

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, TT This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm)-5-Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 ___ 40 ^ 6¾¾ b7 V. Description of the invention () First, rice α-starch hydrolase The cDNAs P0S103 and P0S137 have been studied in detail. The α-amylase precursor protein produced by P0S103 and P0S137 has a molecular weight of about 48 0a. When it is secreted outside the cell, the signal peptide chain of the precursor protein is It is excised so that the mature α-amylase has a molecular weight of about 45 to 46 KDa and its isoelectric point is predicted to be about 6.0. However, Kumagai et al. (1990) transplanted POS103 into yeast cells and allowed the yeast to secrete oc-amylase into the culture medium. It was found that the molecular weight of ex-amylase was about 44 to 4503, and the isoelectric point was about It is 4.7 to 5.0. The secretion mechanism of plant proteins has not been understood so far. It is generally believed that it may be similar to the secretion mechanism of animals. Secretory proteins are first synthesized by ribosomes on the rough endoplasmic reticulum (UER) membrane. The function of the chain guides the extended protein into the inner cavity of the endoplasmic reticulum (ER). After the signal peptide is excised by the signal peptide, the ER forms a vesicle by budding to contain the protein, and then transfers it to Golgi In combination, the protein is delivered to Golgi. In Golgi apparatus, secretory vesicles are transferred to the cell membrane, and proteins are secreted outside the cell after membrane fusion (Walter et al., 1984). N-glycosylation is found in many secreted proteins. It is not clear what role glycosylation plays in protein secretion. It is known that secreted α-amylases in aleurone cells of barley and wheat are not glycosylated (Jacobsen et al., 1988; Tkachuk & Kruger, 1974). Akazawa et al. (1985) have shown that ct-amylase produced by the blastoderm (scute 1 1 um) during budding is a secreted protein that is glycosylated during emergence. However, 〇 'Me U 1 et al. (1990) believed that (please read the precautions on the back before filling this page). Binding-. 4 This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public) (Centi) -6-402638 A7 B7 _ ^ ___ 5. Description of the invention () (Please read the precautions on the back before filling out this page) Mature (X-amylase and at least 2 species produced during rice seed germination) Not glycosylated, POS137 and λ OSgl, respectively, because there is no glycosylation site in the amino acid sequence. Akazawa et al. (1986) found that during the formation of α-amylase, the signal peptide chain was After removal, an oligosaccharide with a molecular weight of 2900 Da will bind to the α-amylase. Treated with an inhibitor of glycosylation, Tunica mycin (tunica my ci η). The blastoderm cells were found to inhibit sugar Abasification does not inhibit the secretion of α-amylase (Akazawa & Nishimura, 1985). Therefore, the secretion of cereal α_amylase may not be related to glycosylation. Using wheat and rice α-amylase genes Transplantation into yeast It was found that wheat α-amylase was not glycosylated and its secretion was not much (Rothste i η et a 1., 19δ7), while rice α-amylase was not only glycosylated and secreted a large amount of ct-amylase (Kumagai et al., 1990), so Kumaga i and others believe that although glycosylation has nothing to do with the secretion of protein, it will affect the secretion of cereal-amylase. The central standard of the Ministry of Economic Affairs The secretion of α-starch hydrolase in the cells of the barley aleurone printed by the Bureau of Work and Consumer Cooperatives is also regulated by calcium ions (Busheta 丨., 1 9 8 9_). Ct-丨 Dianfen hydrolase In the absence of evil ions, α-amylase will lose its enzyme activity and become unstable. GA 3 can regulate calcium ions into the ER lumen and combine with ct-amylase, so GA3 may be It also regulates the post-translational level of α-amylase synthesis (Bush et a 1 ·, 1989) ° Applying gene transfer technology, using single cells as materials, to analyze the expression and function of eukaryotic genes, is to study Other biological gene regulation mechanism This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -7 402638 A7 B7 ---________ ~~: An ideal of the invention description (5) The plant suspension culture cells have the characteristics of fast growth rate, more consistent differentiation, easy to separate protoplasts, convenient operation, and fast response to external treatment. Therefore, it is a study of plant physiology, biochemistry, transmission, breeding, and molecular biology. Very ideal material. In contrast, studying the regulation mechanism of gene expression using a whole plant or a specific tissue of a plant body as a material involves complex physiological and biochemical reactions, so it is not easy to study. In the past two years of research, the inventors of this case have known that the suspension of cultured rice cells can control the expression of certain α-starch hydrolase genes by controlling the presence or absence of sucrose in the medium. In the absence of sugar in the culture medium, the α-amylase gene was stimulated and expressed in large quantities, and the synthesis and secretion of α-amylase were also increased. This case is aimed at this phenomenon. It is expected that the promoter of the α-amylase hydrolysate gene and the DNA sequence of the signal chain of the signal chain will be selected from the DNA bank of the rice gene group to further study the carbohydrates to the α-amylase. The regulation mechanism of gene expression and the pathway and mechanism of α-amylase secretion, and then this new mechanism can be used in the field of engineering technology. Cover, the use of plant cells to make genetic engineering proteins has the advantages of using traditional methods such as bacteria and yeast, for example: printed by the Consumer Cooperative of the Central Procurement Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) 1. Plant cells are higher nucleated cell organisms. The protein produced by them has undergone many complex post-translational modifications to make the protein active, while bacteria lack a post-translational system, and yeast has limited modifications; 2. Baeulovirus is a virus of insect cells. The use of it to produce recombinant proteins usually has excellent activity. Therefore, this system is a commonly used gene expression system. However, the virus must be applied to Chinese paper. National Standard (CNS) A4 specification (210X297 mm) -8 _ 403638 A7 B7 V. Description of the invention () The genetically modified prion protein can only be prepared by staining cultured pygma cells, and the culture medium for insect cells must contain serum, cost Very high; 3. Utilize the newly developed ct-amylase gene expression system in this case, so Into protein synthesis may amount to a sudden increase in the medium under glucose deprivation conditions, even 50-fold or more even, and the protein may be secreted into the culture medium can be simplified purification procedures. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) In the past two years of research by the inventor of the case, it was found that the rice α-starch hydrolase gene family belongs to two different types Regulation: (1) hormone regulation of Langya seeds, and (2) metabolic inhibition by useful carbohydrate nutrients in cultured cells. It is known that the expression of a-amylase hydrolase gene in degermed rice seeds is induced by exogenous gibberellic acid. On the other hand, the expression of α-amylase in floating-cultured cells is induced by a lack of sulfohydrate nutrients. Induction of ot-amylase mRNA in sucrose-deficient media requires a delay period of 2 to 4 hours. The induction of the ex-amylase gene expression was unusually high and the alpha-amylase inRNA level increased 8 to 20 times after 24 hours of sucrose deficiency. The synthesis and secretion of ex-amylase also depends on the level of the master source. Desuppression or inhibition of α-amylase synthesis can be reversed by removing sucrose from the medium or supplementing the sucrose to the medium, respectively. Glucose and fructose also exert an inhibitory effect on ct-amylase synthesis similar to that produced by sucrose. (Su-May Yu et al., &Quot; Metabolic Depression • of a -amylase Gene Expression in Suspension-cultured C e 1 1 sof R ice, "will be published in J. Biol. Chem. Y 〇1 266. Paper size Applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) -9-402638 at _B7___ V. Description of Invention () 1991 Nov .; Su-May Yu et al., &Quot; Catabolic Depression of a -amylase Gene Expression in Suspension-cultured Cells of Rice, "In preparation for publication, this article is incorporated into this case as a reference). Using gene transfer technology to transduce DNA into plant cells has become the most common and best method for studying the regulatory mechanism of genes. Transplanting DNA into plant cells or tissues, in addition to using the method of Agrobacterium Ti plastids (Bevan & chilton, 1982), there are two chemical methods and physical methods. Chemical methods include the use of the PEG method (Kι-ens et al., 1982), the poly-L ornithine method (Davey et al., 1980), and the basic acid recording method (Hain et al. , 19 85) and so on. These chemicals are toxic to cells, so they may cause cell damage and affect transfection efficiency. The laws of physics include the electrical penetration method (Fromm et al., 1986), the microinjection method (Crossway et al., 1986), the particle collision method (Klein etal., 1987), and the ultrasonic method (Joersbo & Brunstedt, 1990), etc., the principle used is to create holes in the cell surface by mechanical force to facilitate the entry of DNA, so it will also cause some damage to the cells. Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling out this page). In addition, the transferred plastid DNA should carry the resistance gene and the reporting gene. The resistance gene can be used as a selective marker for transgenic plants because its products can resist the action of certain antibiotics or drugs. There are two types of resistance genes commonly used in plants, one is the hygromyein B resistance gene (Waldron et al., 1984). It can inhibit the effect of hygromycin. The other is the neomycin phosphotransferase I (NPT E) gene (Herrera et. This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) -10_ 40263S A7 B7 staff consumption of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative printed i, invention description () al., 1983), can inhibit the effects of kanamycin and G418, but many grasses are naturally resistant to kanamycin, so use NPT II As a selectable marker, it may be difficult to determine the success of the transplantation (Hauptmann et al., 1988; Potrykus et al., 1985) ° The reported gene is a gene that acts as a coding region during gene fusion, and its transcription and translation are directly affected by Controlled by genes themselves or other gene promoters (Jeferson, 1987). There are seven more commonly reported genes in higher plants, namely / S-galactosidase UacziKHelmer 'et al., 1984), and chlorimine acetamidine transferase (CAT) (Gorman et al., 1982), Neomycin basic acid transferase (NPT H) (Reiss et ah, 1984), nopaline synthetase (NOS) (Beral et al., 1983), octopus test synthase, (〇CS) (DeGreve et al., 1982), firefly insect photonase (Ow et a 1., 1986) and Escherichia coli / 3-glucuronidase (GUS) Uefferson et al., 1 987). Among the above seven reported genes, the plant itself will produce lacz enzymes, and the analysis of enzymes such as NOS, OCS, and firefly luciferase is more troublesome. At present, the enzyme analysis of the more common CAT and NPT E is also expensive and difficult. Moreover, the esterase, phosphoesterase and transferase contained in the plant itself will compete with the substrates of CAT and NPT E, so it is relatively Ground 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 that strong GUS activity can be measured in the fruit and immature seeds of seed plants, the GUS analysis method is simple and rapid (Please read the notes on the back before filling this page)

The paper size of the edition is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 40S638 A7-A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economy -Chloro-3-indolyl glucuronide (X-gluc) is cheaper, has good GUS stability, and has low GUS activity in most plants (Jefferson, 1987). The GUS gene is the ideal reporter gene for higher plants. In view of the fact that α-starch hydrolase has been widely used in the industry, it is necessary to obtain this enzyme in a large and simple way for the industry. Therefore, the inventors found that the above-mentioned purpose can be achieved by causing the lack of sugar in the medium to induce the expression of the α-amylase enzyme gene. Furthermore, the gene expression system of O-amylase can be used to obtain a large amount of other desired proteins integrated into the gene expression system under the condition of sugar deficiency. Therefore, one of the purposes of this case is to provide a method for generating a large amount of (X-amylase). Another purpose of this case is to provide a novel gene expression system to produce a protein for transmission engineering for mass production of the desired protein. The present invention will be further explained with the following examples and drawings. However, as will be understood by those skilled in the art, the following examples are only used to illustrate the features of the present invention, but not to limit the invention. Materials ΦMethod I 1. Screening of the ct-amylase gene from the DNA bank of rice genome 1. Brief introduction of the DNA bank of rice genome: 1. The DNA bank of rice genome used in this case was purchased from CLONTCH (cat # FL 10 40 D, Lot # 7081) company, the rice variety used is 0 ry za sativa L. Japonica. Genomic DNA is extracted from rice seedlings grown in the dark for 7 days and then made into a genetic DNA bank. (Please read the notes on the back before filling in (This page) Binding. Staple-Tip-This paper size applies to Chinese National Standard (CNS) A4 (210X2.97 mm) -12-402638 A7

V. Description of the invention () 2. The selection vector of this library is EMBL-3, and the selection position is Bam HI '. The average DNA fragment length of the selected gene group is 15kb. 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 deposits ’The host bacteria of this EMBL-3 bank are Escherichia coli NM538 or LE 392, which can produce large lytic plaques on agar culture medium. 1.2 Method for screening α-amylase from rice DNA gene bank: 1.2. 1. Preparation and infection of phage host bacteria: 1. Add 5 ml LB liquid culture medium (per liter containing NaCl 10g, tryptic gland 10g, yeast extract 5g), a single colony of large intestine _ NM538 was picked with a toothpick into this test tube, and cultured at 37 ° C with shaking for about 6 to 8 hours until the ODbdd value was 0.5 to 0.7. 2. Centrifuge at 4 ° C for 10 minutes using a Sorvall RT 6000B centrifuge at 3000 rpm, and dissolve the precipitated bacteria with ice-cold MgS04 (add 3m 1 MgSO 4 to each 10m of the bacterial solution precipitate) Dissolve), store it at 4 ° C for future use (approximately one week) ° Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 3. Dissolve 80ml of Top Agar in a microwave oven (1M MgSCU 10ml per liter of LB liquid medium, 10N NaOH 0.2ml, agar 8g) 'Divide them into 8 tubes, each containing 9ml of Top Agar, and incubate in a water bath at 46 ° C. 4. The 6 «1 rice gene bank (potency 2x10 4" 1) is thoroughly mixed with the prepared 2.4ml HM538_ solution, and cultured at 37 ° C for 15 ~ 20 minutes. 5> Take 300ml of mixed bacteria solution and add this 8 tubes of Top agar gel. The paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) -13-Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 402638 A7 --- -B7 __ 5. Description of the invention () Close the test tube cap tightly, then shake it up and down once to mix thoroughly, and quickly pour the solid medium (1 liter LB liquid medium) which has been pre-warmed at 37 ° C in a large petri dish (diameter 14c! «). Add 15g of agar), and wait for Top agar to gel. After incubation, place a 48 値 petri dish in a 37 ° C incubator and incubate overnight. The next day, you will see dense lysobacteria. 1. 2.2. DNA transfer to nitrocellulose filter paper: 1. Prepare 3 shallow plates, one on each of which is a Whatman 3MM filter paper. The three shallow plate filters are each denatured with a denaturing solution (Ό. 5M NaOH + 1.5M NaCl) and neutralized. The solution (4.5M HaCl + l.OM Tris-HCl, pH 8.0) and 2xSSC (17.5g NaCl and 8.8g sodium acetate per liter) are fully moistened. Wet. 2. Mark the nitrocellulose (NC) filter paper with a signature pen, and cover the solid medium with plaque lightly on the side facing down, and then use a pin to kill four asymmetric points on the medium. For the purpose of picking plaques in the future. 3. Slowly tear the MC filter paper from the culture medium with the side facing up, place it on the denaturing solution for 5 minutes, then transfer to the neutralizing solution for 5 minutes, and finally Reaction in 2xSSC for 2 minutes. 4. Place the NC filter paper on 3MM filter paper, dry it, and bake in an oven at 80 ° C for 2 hours. 1 · 2.3. Use probe hybridization from the gene DNA library Screening of ct-amylase genes: 1.2.3.1. Agar colloid recovery 1) 1 ^ (^ 1311131 ^ 5 61: £ 11., 1989): 1. Rice CC-amylase cDNA pure strain RMAYC30 (Tzou, 1 990 ) This paper size applies Chinese National Standard (CNS) A4 specification (210X2.97mm) -14-(Please read the precautions on the back before filling this page)

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 402638 a7 B7 V. Description of the invention () After limiting the action of EcoRI, add 0.15 times the volume of loading buffer (0.25% bromophenol blue, 0.25% cyanol, 30% glycerol) ° 2. Use agar colloid (0.8% agar_gelatin) in 5] buffer (0.089 Tris, 0.039M boric acid, 0.002M EDTA) to 100 Volt voltage for electrophoresis. 3. The electrophoretic colloid is stained with ethidium bromide (e t h i d i u m b r 0 m i d e), and is placed at a long wavelength (300-360 nm). The position of the desired DNA fragment is determined under a UV lamp, and the colloid at this position is cut off with a blade. 4. Put the colloid in an appropriate dialysis bag and fill it with ΙχΤΒΕ buffer solution. Use the dialysis bag to clamp and clamp the two ends of the bag to avoid air bubbles. Place it flat in the electrophoresis tank. The electrophoresis solution in the tank must cover the bag. . 5. Electrophoresis at 100 V for about 1.5 to 2 hours. Check D N A under the UV lamp. If the gel has run out of the bag into the solution, put it back in the electrophoresis tank and run it with the opposite electrode for about 2 minutes. 6. Carefully recover the liquid in the bag and extract once with ester and chloroform. Add 0.1 volumes of sodium acetate and the same amount of isopropane (IPA), mix well and place at -20 ° C for at least 2 hours. 7. Centrifuge at 4 ° C and 14000 rpjn for 30 minutes, discard the supernatant, wash once with 70% and 100% alcohol, and dry the DNA by vacuum centrifugation. 8. Add appropriate TE buffer to dissolve the dried DNA. 1.2.3.2. Preparation of probes: 1.2, 3.2.1. Random model selling method (1? 611 ^ 0guang8 & ¥ 〇 卩 61316111, 1983): This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X 297 mm) -15-(Please read the notes on the back before filling this page) -ο Install. Order

A7 B7 402638 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5) Description of the Invention (1) Take 100 ~ 200ng of DNA and add sterilized deionized water to make the volume to 5.5 * 11, then at 100 ° After heating in C for 3 minutes, it was quickly inserted into ice to allow it to cool rapidly. 2. Add lwl BSA (l0wg / iil), 1.5wl hexamer (25ng / wl), lOiil in 2.5x reaction buffer (0.5M Hepes, pH 6.6; 12.5mM MgCla; 25mM / 3-S1-based ethanol; 125mM Tr > is, pH 8.0, 50mM dATP, 50mM dGTP, 50wM dTTP), 5m1 [a-32P] dCTP (10iici // ul, ljul Klenow enzyme) (BRL, 5 units / mI) and mix well. 3. Allow to stand for at least 2 hours at room temperature. 4. Add TE buffer to increase the volume by 200W 1. 5. Extract once with phenol and chloroform. Add 20mI NaOAcxSitI tRNA (20u.g / < il), 200jnl IPA, mix well and place at -20 ° C for at least 2 hours. 6. Centrifuge at 14000 rpm for 20 minutes. Aspirate the supernatant, wash the precipitated probe DNA twice with 75¾ alcohol, and dry the DNA by vacuum centrifugation. 7. Take 0.5 · 1w of the probe solution and measure its cpffl using a flash counter to estimate the amount of probe solution required for hybridization. Probe solution volume (/ 1 1) = 2 ~ 5xl05cpm / ml X hybridization reaction solution volume (ml) + probe solution radiation volume (cpm / ju 1) 1 '2. 3 · 2. 2. Melton et al., 1984): 1. This method uses pBlueseriptH KS (+/-) as the carrier, and uses this paper size to apply China National Standards (CNS) A4 (210X 297 mm) -16-(please first (Read the notes on the back and fill out this page)

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40263S A7 i 'invention description () (Please read the notes on the back before filling out this page) It contains the characteristics of the T7 promoter and T3 promoter. T7 RNA polymerase or T3 RNA polymerase is added to the test tube to make a single The counter-intention of RN Α was the probe. In this experiment, this reaction was used to prepare specific gene probes (Tzou, 1990) in order to screen the promoter of the cc-amylase gene in rice and determine four 0-amylase cDNAs (RAMYC26, RAMYC27, RAMYC28, and RAMYC30) may be derived from the α-amylase gene DNA we isolated. 2. Take about 200 ~ 300ng of DNA, add DEPC H2O to make the volume 6 Mi, and then add 5m1 5x translation buffer, U1 10mM rATP, 1 M1 10mM rCTP, 1al 10mM rGTP, 2wl 1mM rUTP-1 μ. 1 0.75M DTT ^ 1m1 RNase-block (40U / wl RNasin), 2 * U yeast t-RHA (10a1 / m1), 5ju1 a-32P-UTP (Amersham, 10Wci / ul), lwi T7 or T3 RNA polymerase After mixing, react at 37 ° C for 45 minutes. 3. Add 20ul DEPC Hs0, 5 / il DNase I buffer (10x), 0.5wl DNase I (23u / Wl), mix well, and react at 37 ° C for 15 minutes. 4. Same method as 1.2, 3. 2. 2.1. Steps 5, 6, and 7. Printed by a member of the Central Standards Bureau of the Ministry of Economic Affairs and a Consumer Cooperative. 1.2.3.3. Probe hybridization: 1. Soak the dried NC filter paper in 5xSSC. 2. Transfer this NC filter paper to an appropriate amount of pre-hybridization solution (50¾ formamidine, 0.7 5 M Na C 1,50 m Tris, p Η 7.5, 0.1% Na 4 P 2 07, SDS, lmM EDTA, 1x Denhardt's solution, salmon sperm DNA). Pre-hybridization solution (m 1) = (0.04) This paper size is applicable to Chinese National Standard (CNS) A4 specification (21〇 × 297mm) -17-4026S8 Α7 Β7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Description of the invention () «11 / ^ 114 per piece} &(;: area of filter paper ((: 1112) \ ^ (: number of filter paper). Salmon sperm DN A needs to be heated to 100 ° c for 5 minutes.) , And then quickly put in ice to separate the double strands of DNA before use. 3. Shake the reaction in a constant temperature box at 42 ° C for 3 hours. 4. Heat the probe containing the α-amylase gene to 100 After 3 minutes at ° C (RNA probe does not need to be heated), quickly insert it into the ice. After it is cooled, add it to the pre-hybridization solution, mix well, and then put it back into the 42 ° C incubator to react overnight. The hybridization reaction solution was discarded, and the washing solution (0.1xSSC, 0.1% SDS) was shaken and washed twice at room temperature, and then shaken and washed at 50 ° C for 30 minutes. The radioactivity was measured by a Geiger counter. If it is too high, wash it with a washing liquid at 55 ° C or higher for 30 minutes. 6. Dry the NC filter paper, and then place the four asymmetry on the NC filter paper. Each spot is doped with a trace amount of (cc -35S) dATP ink, so that the negatives can be used as a reference for orientation identification after the film is washed. 1.2.4. Purification of pure strains of the amylolytic enzyme gene group: 1. Initially obtained from the previous steps It has a hybrid positive lysozyme, and this lysozyme contains the α-amylase enzyme gene. Because the hybridization reaction obtained from the exposed negative film may contain several lysolytic plaques, these lysolytic plaques need to be excavated for more It is further determined that the maggot really has a positive hybridization reaction, but the phage concentration of these plaques may be too high, so it is necessary to serially dilute and then select an appropriate concentration for probe hybridization. 2. After preliminary hybridization, use the tip of a pipette. The paper size included in the excavation of the hybrid positive reaction point is applicable to the Chinese National Standard (CNS) A4 specification (210X2.97 mm) _ 18-(Please read the precautions on the back before filling this page)

Order the printing machine of the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs _ A7 ^ ~ _B7_; __1 'Description of the invention pH 7.5; 100 mM NaCl, 10 mM MgS04), 20wl of chloroform were mixed thoroughly. 3. Let stand for 2 hours at room temperature to allow the phage to diffuse out of the agar gum. On average, there are 108 to 107 phage particles per lysozyme. 4. Bacteriophage serial dilution method: Prepare three 1.5ml 1 small centrifuge tubes, add 100wl, 180W1, 90 * il phage dilution buffer, and then take 1 «1 and 1000 / Π from the phage stock solution in step 2. The phage dilution solution is mixed, which is 10-3 times the phage dilution solution. From the 10-3 times solution, 20w 1 and 180 w 1 of the dilution solution are mixed, that is, the dilution is 10-4 times, and then 10 μ1 and The dilution solution of 90; il is mixed 10 to 5 times, so a phage dilution solution of 10 to 3, 10 to 4, and 10 to S can be obtained. 5. Take lOOwl of NM 538_ solution (0D60O = 0.5 ~ 0.7) to 3 値 clean eppendorf test tube, and then draw 10.3, 10'4, 10_ 5 times the phage dilution solution 100w each and Mix the solution and incubate at 37 ° C for 15 minutes. 6. Take 3 5ml test tubes and add 3ml of melted Top Agar and 30ml of 1M MgCl 2 each, and keep them in a water bath at 48 ° C. 7. Mix the mixture of phage and bacteria with Top agar evenly, quickly pour it onto LB solid medium (diameter 9cm) pre-warmed at 37 ° C, and spread evenly. After the Top agar has coagulated, it is at 37 ° C. Incubate overnight. 8. Repeat steps 1.2.2., 1.2.3., And 1.2.4. Until the medium contains the paper size, the Chinese National Standard (CNS) A4 specification (2! 〇χ297 mm) -19-(Please read first Note on the back, please fill out this page again) © _ 装. Order 402638 A7 A7 B7 V. Description of the invention () 0C-Starch hydrolase-based gene lysozyme is only derived from a single lysozyme. I. Analysis of pure strains of rice α-amylase gene group: 2. 1. Purification of phage DNA: 1. Inoculate host fine NM 538 in 10 ml of LB + Mg * 2 liquid medium (10 ml of 1 M MgS per LB. 4) Incubate at 37 ° C with shaking overnight. 2. Add 500 ml 1 LB + M g + 2 medium to a 500 ml 1 Erlenmeyer flask, and then culture it with 4 ml of host bacteria and 5 to 10 lysate (or 500w 1 The phage stock solution) was warmed and cultured overnight at 37 ° C with shaking. 3. Bacterial corpse eroded by phage will be seen in the culture medium overnight, so the medium is slightly clear. At this time, 5W 1 of DNase I (10wg / Ml) and RNase (10Wg / Wl) can be added. Incubate with shaking at 37 ° C for 30 minutes. 4. Use a GSA rotor in a Sorvall RC 5C centrifuge at 4 ° C and 8000 rpm for 10 minutes. The upper clear solution is the phage solution. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) 5. In a Beckman centrifuge tube (Cat. No. 344058), add 6ml cushion solution (40% glycerol, 20mMMgS〇4 , 50 mM NaCl, 10 mM Tris-HCl, pH 8.0), and then slowly inject the phage solution, until S is 0.5 cm from the nozzle. After equilibration, centrifuge in a Beckman Sw28 rotor, LB-M ultra high speed centrifuge at 4 ° C and 27000 rpm for 1.5 hours. This paper is again applicable to China National Standard (CNS) A4 specifications (210 X 297 mm) -20-A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 402638 i. Description of invention (6) After centrifugation, discard the upper layer Liquid, take the centrifuge tube upside down, use scissors to cut off near the bottom of the tube, cover the centrifuge tube to avoid contamination by other debris. The precipitated phage was dissolved in 150W 1 ΝΤΕ buffer solution (300mM NaCl, 100mM Tris-HCl, pH 8.0, 1mM EDTA), transferred to an eppendorf tube, and the remaining phage was precipitated once with 150m 1 ΝΤΕ buffer. 7. Add the same amount of phenol to extract twice, and then extract again with chloroform. 8. Add 30wl 4.4M ammonium acetate pH 5.2, 350 * 11 isopropanol and mix well, and then the phage DNA precipitation can be seen. Centrifuge at 14,000 prm for 5 minutes in an eppendorf centrifuge at 4 ° C to obtain phage DNA. 9. Wash the precipitated phage DNA once with 70¾: alcohol, then once with 100% alcohol, and vacuum dry the DNA. 10. Add 40wl of HE buffer (10mM Tris, ImM EDTA) to dissolve the dried DNA, and take 1w1 of DNA solution for restriction analysis. 2.2. Southern blot analysis: 1. After bacteriophage DNA acts to limit the enzyme, add 0.15 ploid loading buffer (0.25% bromophenol blue, 0.25% xylaniline '30¾ glycerol) ).

2 · Use agar colloid (0.8% agar gel) in TBE buffer (0.089M

Tris, 0.089M boric acid, 0.002M EDTA) and electrophoresis at 100 volts. 3. After electrophoresis, the agar colloid is stained with ethidium bromide, and the photo is taken under the U V lamp. (Please read the precautions on the back before filling in this page)

This paper size applies to Chinese National Standard (CNS) A4 specifications (210X297 male 1 :)-21-Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (4) Agar colloid with denatured solution at room temperature Soak for about 1 hour with shaking, and shake with neutralizing solution for 1 hour. 5. Cover the bridge made of acrylic plate on a shallow plate, and cover it with a 3MM filter paper, so that it stretches under the bridge so that the solution in the shallow plate can be sucked. Then cover it with a 3MM filter paper of the same size as the bridge. Pour some lOxSSC solution on the filter paper to make the filter paper wet, and then place the colloid on the filter paper to flatten the gene sieve membrane wetted in 1 OxSSC Cover the gel, remove any air bubbles, and cover it with two pieces of 3MM filter paper moistened with 10xSSC. 6. Pour an appropriate amount of 1 OxSSC solution into a shallow dish, cover the uncovered area around the bridge with a two-layer cleaning film, and then place a stack of absorbent paper on the filter paper above the colloid. Press lightly on the book overnight. 7. Take out the gene sieve analysis membrane and bake in an oven at 80 ° C for 2 hours. 8. Pack the baked gene screening membrane in a hybridization bag and add 2xSSC solution to moisten the gene screening membrane. After squeezing the excess 2xSSC solution, add an appropriate amount of the 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. Selection of DNAH segment of rice ex-amylase gene group: 2. 3. 1. Preparation of appropriate cells (competent cel 1) (E. coli XL1-B strain): 1. Pick XU- A single colony of B was inoculated into 5 ml of LB liquid medium and cultured overnight at 37 ° C with shaking. 2. Pour 5ml of bacterial solution into 500ml of LB medium. Shake the culture paper at 37 ° C to apply Chinese National Standard (CNS) A4 specification (210X 297mm). (Please read the precautions on the back before filling this page. )

Printed by the Employees' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of Invention () _ Raise to 0 D 5 5 0 = 0.3 5 to 0.5. 3. Centrifuge at 4 ° C and 5000rpm for 5 minutes. 4. Discard the supernatant, place the centrifuge tube in a water bath, and dissolve the precipitated body with 200 ml of ice-cold 0.1 M MgC 1. 5. Centrifuge at 5000 rpm for 5 minutes, lyse the cells with 200 ml of ice-cold 0.1M CaCle, place in an ice bath for 20 minutes, and then centrifuge at 5000 rpm for 5 minutes. 6. Add 43 «11 ice-cold 0.1 ^ € 3 (: 12 to dissolve the bacteria, then add 71111 pre-cooled glycerin, and mix well in the ice bath. 7. Dispense the« solution into an Eppendorf test tube and place Liquid nitrogen was rapidly frozen and then stored in a refrigerator at -70 ° C. 2.3.2.2. Conjugation reaction: 2.3.2.1 Dephosphorization of linear DNA: 1. Using a single restriction enzyme, IO / The IG plastid PBlueSCHpt] I KS (+ /-) (Stratagene) was cut open, and TE buffer was added to make the I volume become 100ju 1.2. According to the method of recovering DNA by agar colloid (section 1.2.1.1. Steps 6, 7, and 8 of this section can obtain purified linear proton DN A. 3. Add 20 < il TE buffer to dissolve the DNA, and then add 2 units of CIAP (calf intestinal alkaline phospholipase) and 2 ml 1 10xC IAP dephosphorylation buffer (10 mM ZnCla, 10 mM MgCIa, 100 mM Tris), and incubate at 37 ° C for 30 minutes. 4. Same as step 2. 5. Repeat steps 3 and 2 again. This paper scale applies to China National Erase Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

402638 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs i'Invention Note (6) Add 20w 1 TE buffer to dissolve the dephosphorylated plastid DNA and store at 4 ° C. 2.3.2.2. Conjugation of sticky-end DNA: 1. Please refer to the method of agar colloid to graze DNA (steps 1, 2, 3 in section 1.2.3. 1.). However, the preparation of colloids was changed to low melting point Nus ieve agar (FMC, # 10259), and 5 ttl of ethidium bromide (10 »g / Al) was added per 100 μm of agar colloid. 2. On a 1JV light, cut the DNA colloid and place it in a 68 ° C water bath for 10 minutes to melt the colloid and incubate in a 37 ° C water bath. 3. Take 20.5wl of DNA, add 0.5wl of dephosphorylated plastid DNA (0.2 wg / M), 6 # 1 5xT4 DNA ligase buffer (100mM Tris, 25mM MgCls, 25mM DTT, 25〇wg // il BSA) > 3iil T4 DNA ligase (1 unit / ill). 4. Reaction in a water bath at 16 ° C overnight. 2. 3. 2.3. Conjugation of homozygous DNA: 1. Use the method of recovering DNA by agar colloid to obtain purified D N A of sticky ends. 2. Take 40m1 DNA, add 2 / il 2mM dNTP mix, 3 / ul Klenow enzyme (5u // il), 5a1 lOx fracture translation buffer (0.5M Tris, 0.1M MgS04, ImM DTT, 500 * ig / ffll BSA), mixed well, and reacted at room temperature for 30 minutes, then the sticky ends of DNA can be patched up. 3. Add 2μ1 0.5M EDTA, and then extract once with tritium and chloroform, repeat the method of recovering DNA from agar colloid (the steps in section 1.2.3.1.1. This paper size applies Chinese National Standard (CNS) A4 specification (210X297 (Mm) (Please read the notes on the back before filling out this page) Binding and binding. 4 A7 B7 Employee Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs _ System V. Description of the invention () 6 '7, 8) ° 4. 43.5 μ1 DNA, plastid DNA (0.2 Mg / wl) with 0.5 * U blunt ends, IOjuI 5x · T4 DNA ligase buffer, 6 from 1 T4 DNA ligase (iil / ml) were added. 5. Reaction in a water bath at 16 ° C overnight. 2.3. 3. Bacterial transplantation: 1. After the rice α-amylase gene group DNA fragment and plastid DNA are ligated and reacted overnight, if they are reacted in colloids with low melting point, they are heated to 68 ° C 5 Minutes and incubate in a 37 ° C water bath. 2. Add 200 * 11 appropriate cells (XL11ue) and place in an ice bath for 30 minutes. 3. Quickly place in a 42 ° C water bath for 3 minutes. 4. Add 100wl LB medium and incubate at 37 ° C with shaking for 30 minutes to 1 hour. 5. In the LB solid medium containing ampicillin iSOiig / nil), each coated with 75wl X_gal (2%, 5-bromo-4-chloro-3-, late-yl-fluorene-DU galactoside and lO. wl IPT.G (100 mM isopropyl-Z3-D-sulfur-B galactopyranoside), and let it stand on a sterile workbench for more than 15 minutes. 6. Apply _ solution to LB solid containing X-Gal and IPTG Cultivate overnight at 37 ° C on culture medium. 2. 4. Purified fine plastid DNA: 2.4. 1, small amount purification method: 1. Pick a single colony and culture in 5 ml LB + Amp (50ug /; ul ) The liquid medium was cultured at 37 ° C overnight with shaking. (Please read the precautions on the reverse side before filling out this page) © Packing _ Binding paper size: Chinese National Standard (CNS) Α4 size (210X297 mm) 402638 A7

Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 2. Pipette 1.5ml of the bacterial solution into an eppendorf test tube and centrifuge at 4 ° C and 14000rpm for 3 minutes. 3. Aspirate the supernatant and freshly prepare ice-cold solution I (50 mM glucose, 10 mM EDTA, 25 mM Tris-Cl ·, 4 mg / ml lytic enzyme) at 100 W to dissolve the precipitated bell body, mix well, and The reaction was performed at room temperature for 5 minutes. 4. Add 200wl of freshly prepared solution I (0.2M NaOH, 1% SDS), mix well and react in an ice bath for 10 minutes. 5. Add 150tfl ice-cold solution] I (5M CH3C00K, ρΗ4.δ), mix well, and react in an ice bath for 10 minutes. 6. Centrifuge at 4 ° C and 1000 rpm for 10 minutes. 7. Aspirate the supernatant and extract once with phenol and chloroform, add the same amount of isopropanol, and place in the refrigerator at -20 ° C for at least 1 hour. S. Centrifuge at 4 ° C and 1400 ° · ριη for 30 minutes, discard the supernatant, wash each with 70% and 100¾ alcohol, and dry the DNA by vacuum centrifugation. 9. Add 16.8μ1 TE buffer to dissolve DNA. 10. If this DNA is used for DNA sequence analysis, then add 3.2m 1 5M HaCl, 20wl 13% PEG (8000), and mix in an ice bath for at least 30 minutes. 11. Repeat step 8 to dissolve the DNA in 16m 1 TE buffer and store at 4 ° C for DNA sequence analysis. 2.4.2. Mass purification method: 1. For 5 ml of the bacterial solution cultured overnight, add 250 ml of containing ampicillin (50 This paper is again applicable to the Chinese National Standard (CNS) A4 specification (210X2.97) Li) (Please read the precautions on the reverse side before filling out this page) te. Order--, 4- Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention ()

Wg / ml) in 2xYT medium (16 g per liter of bacterial trypsin, 10 g of yeast extract, 5 g of NaCl, 0.5 ml of ION NaOH), and cultured overnight at 37 ° C with shaking. 2. Centrifuge for 5 minutes at 45,000 rpm with a GAS rotor. 3. Discard the supernatant * and add 5ml of solution I (25mM Tris-Cl, 10mM EDTA, 15% Sucrose, 2mg / ml lysozyme) to lyse the bacteria '. Transfer the bacteria to a 40ml Oakr idge test tube, The reaction was carried out in an ice bath for 20 minutes. 4. Add 9.5nrl solution E (0.2M NaOH, 1¾ SDS), gently shake the centrifuge tube up and down to mix well, and react in an ice bath for 10 minutes. 5. Add 7.5 Inl solution IlI (3MCH3C00Na, pH 4.8), mix gently and place in an ice bath for 20 minutes. 6. Centrifuge in SS34 rotor for 30 minutes at 12,000 rpm. Filter the supernatant with two layers of gauze or one layer of mirde loth, add equal amounts of isopropanol, and place in a refrigerator at -20 ° C for at least 2 hours. 7. Centrifuge at 12,000 rpm for 30 minutes, discard the supernatant, wash the precipitated DNA once with 10025 ethanol, and dry the DNA by vacuum aspiration. 8. Dissolve the DNA in 4.2ml TE buffer, add 5wl RNase (10 mg / ml) and let stand at 37 ° C for 20 minutes. 9 · Add 4.5 g of C s C 1 and gently shake to completely dissolve. Then transfer the DNA to a Quick Seal tube (Beckman, 5.lml, No. 344075) with a dropper. 10. Fill the centrifuge tube with ethidium bromide (10 mg / in 1). After equilibration, seal the mouth of the tube with τ u b e Sealer (Beckman). This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

402638 A7 B7 V. Description of the invention 11. Use VTi 65.2 rotor. Centrifuge at 20 ° C and 55000rpm for 15 hours. 12. Under a long-wavelength UV lamp, use a needle to extract plastid DNA, and then extract the red ethidium bromide several times with butanol until the D N A solution is clear and colorless. 13. Perform dialysis in a dialysis bag in TE buffer for about 12 to 20 hours to remove CsC 1 during which the TE buffer needs to be changed about three times. 14. Collect the DNA solution and measure the DNA concentration using a spectrophotometer. I. DNA sequence analysis Promoter of rice α-amylase gene: 3. 1. 5 'end of α-amylase gene which is gradually shortened (Hen i kof f, S., 1984) 1. This experiment The principle is to cut two kinds of restriction enzymes at two different parts of DNA, cut out the protruding 3 ′ end at one site, and cut the protruding 5 ′ end at the other part, and then use the exonuclease m (Εχ〇 m) The principle of specific cleavage at the protruding 5 'end or the head with the same length. After taking samples at different reaction times, a series of gradually shortened white horse DNA can be obtained. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page) 2. Take 15ug of DNA to restrict the enzymes Bam HI and Bstx I at 37 ° C for 2 hours. 3. Method for recovering DNA by repeating agar colloid (Sections 1.2, 3.3, Steps 6, 7, 8) ° «· 4. To dissolve the DNA, add 2W1 ΙΟχ'ΕχοΙ buffer solution (660mM Tris pH 8.0, · 6.6mM MgCls) and kept in a 30 ° C water bath. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X2, 97 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 402638 A7 B7 V. Description of the invention (5) Preparation of SI mix (59,5wl H2〇, 9.5m1 7.4xSl buffer, 50 units of SI nuclease), 7.4xSl buffer (0.3M CHaCOOK, pH 4.6, 2.5M NaCl, 10mM ZnSO4, 50¾ glycerol). 6. Dispense 7.5ial SI mix into 8 値 eppendorf tubes and keep in ice bath. 7. Add 400 units of Exo JI enzyme to the DNA sample in step 4. Mix quickly and place in a 30 ° C water bath to react. 8. Take out 2.5ml of the reaction solution S1 mix in the ice bath at one-minute intervals and mix thoroughly, and allow to react at room temperature for 30 minutes. 9. Add U1 S1 Stop Buffer (0.3M Tris base, 0.05M EDTA>), mix and heat at 70 ° C for 10 minutes. 10. Take 2/11 of each DNA sample at each reaction time point for agar colloid. Electrophoresis analysis. 11. Add 1. 5. w 1 K 1 e η 〇wmi .X (1 5 μ) · 1 XK 1 e η 〇w buffer, 5 units of Klenow enzyme), place at 37 ° C to react 3 After 1 minute, add U1 dNTP mix (0.125 mM each of dATP, dCTP, dGTP and dTTP) and react at 37 ° C for at least 5 minutes. 12. Add 40W1 ligase mix (395jul H20, 50m1 10x ligase buffer, 50iU 50% PEG, 5wl 500mM DTT, 5 units of T4 DNA ligase), and react at room temperature for at least 1 hour. 13. Repeat method 2. 3. 3. Steps 2, 3, 4, 5, 6 0 of bacterial transfection 3. 2. DNA sequence analysis: Sanger's DNA-termination method, using Sequenase This paper size applies to Chinese National Standard (CNS) Α4 size (210X297 mm) l ·, -I ------ D Pack II (Please read the precautions on the back before filling this page) < δτ

40863B A7 B7 V. Description of the invention () kit (USB, Version 2.0, No. 70496), synthesizes new strand DNA, and uses U-35S) dATP to calibrate nucleotides. 3.2.1. Preparation of colloid: 1. Wash the glass piece with detergent and wipe with 95% alcohol. After drying, the inner surface of the upper glass piece is wiped evenly with 5% dichlorodimethylsilane once. , Make sure it is smooth, and then fit the glass piece with Spacer '(Reference heart 11131: 13 61: 31,1989, Figure 13.8). 2. Take 48ml Urea mix (containing 420g urea, ΙΟχΤΒΕ 100 ml per liter), 36ml propylammonium stock solution (193g per liter of propylammonium, 420g of urea, 6.7g of bis-propylamine, 10x TBE 100ml) and 60 liter 10¾: Ammonium persulfate, mix well and place in an ice bath. 3. Take 10 ml of the mixed solution and add 20 ml of TEMED (N, N, N, N, -tetra-methyl ethyl amine). After mixing, use _ P ipe 11 ema n_ tip to seal along the Spacer between the glass pieces quickly. Side, clamp the periphery of the glass with a clip, and let it stand for about half an hour. 4. Add 57.5wl TEMED to the acrylamide mixture and mix well. The prepared glass flake colloidal model was tilted so that the bottom corner was at a 45 ° angle with the horizontal, and the propylamidamine mixture was sucked with a 25m 丨 straw using PiPet-aid, and it was slowly injected from the lower hypotenuse. 5. After being filled, the glass piece is slowly laid down in a flat manner, and the top pad is made of Polyuron board so that the top is about 5cm higher than the bottom, and then the flush end of Shark's tooth comb is inserted from the top mouth to about 0.5 cm. . 6. Clamp the top with a clip and let it stand for more than 2 hours. If you want to leave it overnight, seal the top with a cleaning film to prevent the gel from drying. This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) -30-(Please read the notes on the back before filling this page) 40f3638 A7 printed by quasi-station employee cooperatives ______B7_ V. Description of invention () 3. 2.2. Analysis of double-stranded DNA sequence: 1. Take 16m1 supercoiled double-stranded DNA (UgAU) and add 4wl 2M NaOH, mix well at room temperature React for 5 minutes. 2. Add U1 template (10ng / wi), 6wl 3M CHaCOONa and 80 «1 100% alcohol, mix in a refrigerator at -20 ° C for at least 2 hours. 3. Centrifuge at 14,000 rpm for 30 minutes. Wash the precipitated DNA once with 70% and 100% alcohol, and dry DΗA by vacuum centrifugation. 4. Take 4 eppendoff test tubes, add 2.5jal ddATP, ddGTP, ddTTP, and pre-warm them in a 37 ° C water bath. 5. Take 8wl Hs〇 to dissolve the DNA, and then add 20 * Π Se <} uen.ase reaction buffer, liU DTT, 2 «1 diluted mix (0.4 / U dGTP labeled mix + 1,6jul Hb〇 * l, ul («~ 35S) dATP (lO / ici / zul), 2jul diluted sequenase (0.3 < il Sequenase + 1.7wl enzyme dilution buffer), refrigerate under room temperature for 5 minutes. 6. Take 3.5m each. The radiolabeled DNA reaction solution is thoroughly mixed with pre-warmed 2.5 / il ddATP-ddGTP ^ ddCTP, ddTTP, and reacted at 37 ° C for 5 minutes. 7. Add 4w 1 stop solution to each tube. After heating at ° C for 5 minutes, it is quickly placed in Bingyu, and after cooling, it can be analyzed by electrophoresis. 3.2.3. Single strand DN A sequence analysis: The principle of this experiment is to use pBluescriptS KS '(+ /-) plastid with f 1 (+ /-) phage replication initiation point. When the plastids are replicated, through the infection of the phage, the positive strand [SK (+), including the translation of the / 3 -galactosidase gene (please read the back Note: Please fill in this page again.) Order. Qin this paper size is applicable to China National Standard (CNS) A4 (210X2.97mm) -31-Staff Consumption Printed by Zhuosha A7 --..__ B7 __ V. Description of the invention () Cryptographic unit] and minus [(KS (-), including untranslated cryptographic unit] can be integrated into the phage genome. Therefore, the phage is purified DNA, using templates from different directions (KS or SK model dealers), can perform bidirectional femoral DNA sequence analysis. 1. Pick a single colony and add 2xYT + ampicillin (75iig / ml) to the liquid medium. 2 Add 100 ~ 103Pfu / ml of phage generation 3113 (Stratagene, N 0.20 2 51) or R 4 0 8 (S tr * atagene, N 0.2 2 0 2 5 2), in Shake for 2 hours at 37 ° C. 3. Add kanamycin (70 / ig / wl) to the medium of VCS-M13, but not add R408 medium. Shake and culture at 37 ° C for 10-12 hours: 〇4. Take 1.5ml of bacterial solution and centrifuge at 14000rpW for 5 minutes. 5. Pipette 1ml of supernatant > Add 150m 1 PS buffer (20% PEG, 2.5M NaC 1), mix well and keep on ice. 30 minutes in the bath. 6. Centrifuge at 14000 rpm for 20 minutes, aspirate the supernatant, and dissolve the precipitated phage particles with 400jui SE buffer (0.3M CHsCOONa, ImM EDTA). 7. Extract each time with phenol and chloroform, add equal amount of isopropanol, and place at -20 ° C for about 1 hour.

8. Centrifuge at 14000r > pm for 30 minutes, aspirate the supernatant, wash the precipitated DNA with 70% and 100% alcohol each. * Dry the DNA with vacuum centrifugation U1 DMS0, 2 from 1 paper size using Chinese National Standard (CNS) A4 specification (210X297 mm) -32-(Please read the precautions on the back before filling this page)

A7 B7 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention () Sequenase reaction buffer, mixed sentences _. 10. The remaining steps are the same as steps 4, 5, 6, and 7 of the double-strand DNA sequence analysis. However, each tube in step 4 needs to add 0.25 "DMS0. 3.2.4. Computer analysis of DNA sequence: by GCG's sequence analysis computer software (the Genetics Computer Group, University of Wisconsin, Version 5.0, June 1987) (Devereux et al., 1984) for computer analysis of DNA sequence. IV. Transplantation of cells in suspension culture of rice-using electro-penetration method: 4.1. suspension culture of rice: 1. 25ml of 3% sucrose contained in 125ml triangle flask MS (Appendix 1) liquid medium, add 0.5ml 1 suspension cultured rice (Tai Nong 67) cells (about 0.2 g). 2. At 25 ° C, 12 hours light and 12 hours dark, 1.20 rpm speed Shake culture. 3. Subculture is performed every 7 days. 4.2. Transgenes are transfected into rice cells by electro-penetration. 1. Rice suspension culture cells on the third day of growth are taken and the triangles are transferred. The culture solution in the bottle was blotted dry, washed once with the washing medium (Appendix II) CPW7.4, and the cells were transferred to a petri dish * 5m 1 enzyme solution (each 100ml CPW7.4 contains 4% cellulase RS, _1 % m_acerozyme R-10) react at room temperature for 30 2. Collect the enzyme-treated cell solution, transfer it into a 15m 1 centrifuge tube and centrifuge at 800 rpm for 3 minutes. 3. Remove the supernatant * After the precipitated cells are washed twice with CPW7.4, please read the first Please fill in this page for further information.) Packing. ❹ The size of the paper is applicable to Chinese National Standard (CNS) A4 (210X297 mm) -33-Printed by A7 s B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ————— V. Description of the invention () Wash once with EPR buffer (10% glucose, 4mM CaCls, 10mM Hepes pH 7.2) 4. Add 5fii 1 EPR buffer to suspend the washed cells, then add 20 Jug / ml α-amyl hydrolase gene promoter-GUS gene fusion vector to be transduced (PYE132, ΑΕ132, ...) and 10Wg / ml fresh fish sperm DNA, mix evenly at room temperature Incubate for 1 hour. 5. Take 0.8 m 1 of the cell suspension and place it in 0.4 cme 1 ectr Odegap Gene Pul ser cuvette, and leave it in the ice bath for 15 minutes. 6. Place the cuvette in the Gene Pulser (BIO ^ RAD), set the mold (Vo) to 150 volts, the capacitance halo (Co) to 960uF, the resistance ( Ii) 400 ohms, and mold time is 120msec. After the setting is completed, the electrophoretic DNA is transferred to the cells. 7. Place the chilled maggots in the ice bath for 15 minutes. 8. Transfer the cells to a petri dish with 24 wells (Costar, Ho. 3424), add 3 ml 1 KPR (Appendix III) liquid skull culture medium, seal the periphery of the petri dish with paraffin film, and 50 r > pnl culture for 3 days. 4.3. GUS analysis: Preparation of 4,3.1.0 (color reaction solution with -8 111 anti-solution): 1. Dissolve 5mg X-glu in 100 / U DMS0. 2. Add 10 ml reaction buffer (10 μM NaJDTA, 100 μM NaH2P04.H2O, 0.1% Triton, pH 7.0). 3. After mixing the sentences, take 50wl each into eppendorf test tubes. This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) -34-(Please read the precautions on the back before filling (This page).. Βτ 丨 line [A 7 __________B7__ 5. Description of the invention () Stored in a refrigerator at -20 ° C. 4.3.2. GUS analysis: 1. After 3 days of culture of the transfected cells, remove the culture medium, add 50W 1 of X-g i u reaction solution, and leave it in the dark room for 3 hours to react. 2. Remove the cells and observe the staining of the cells under a microscope.结果 Credit results I. Analysis of pure rice a-yodoban hydrolase gene group DNA strains: A pure rice α-starch hydrolase CDAN strain and RAMYC30 coding region were used as probes to screen the rice gene group DNA bank. A total of 16 were obtained. Pure strains of each gene group. The restriction enzyme "Sal I" was used to isolate the PNA autophagosome DNA of the rice gene group. The electrophoresis (see Figures 1A, 1C, 2E) showed that these gene group DNA was cut into several segments. Among them, RAMYG28 and RAMYG18 may have the same electrophoresis picture, and may be the same pure strain. RAMYG17 and RAMYG19 may also be similar pure strains. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs in order to determine the obtained gene group in the DN A bank. Those are the pure strains that contain the rice α-amylase gene. After separating the DNA of Figures 1A, 1C, and 2E on agar gel electrophoresis, the DNA was transferred to the gene screening membrane by Southern blot method, and then The probes made at the 5 'end of the RAMY30 coding region or RAMYC28 coding region were hybridized. The results (Figure 1B, 1D, 2F) showed that RAMYG10, RAMYG11, RAMYG14, and RAMYG20 did not react positively, so it is inferred that this 4 値 pure strain May not contain alpha-amylase enzyme gene * so a total of 12 pure strains of alpha-amylase gene were obtained. Figures IB, ID, and 2F show that some pure plants have 2 to 5 positive hybridization reactions, because this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -35-A7 B7 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the cooperative. V. Description of the invention () It is speculated that the α-amylase gene contained in these pure strains may be divided into several segments by Sa l I, or more than one α-amylase gene on the same DNA (Huang et al, 1990a). 2. A: Determine the 5 'end of the α-amylase gene; In order to identify the pure rice strains that have the 5' end of the α-amylase gene in order to isolate the promoter site, so clean Body, external-translation method, get RAMYC2S and! The 3 'ends of the coding region of iAMYC30 are 320b and 430b in length, plus the 3' end of HS501 and 350b in length (Yu et al, 1990). Through molecular hybridization results (Figure 2B, 2D, 2F, 2H), it was found that RAMYG6, RAPG8, RAMYG17, RAMYG18, RAMYG21 and RAMYG28 all have positive hybridization reactions, showing that these pure strains may contain the "-amylase enzyme promoter site. . Other pure strains may also contain some ot, amylolytic enzyme groups. Because of the 5 'end, it may have low homology with the DNA sequence of the probe used in this experiment, so it does not show positive and negative. 0 RAMYG6, RAMYG28 In the pure strains of the two slugs, two DHA fragments showed positive reactions (Figure 2F). Therefore, it is inferred that the probe used in the experiment (RAMYC28-5 '), in which the DNA sequence may be located at the junction of the two DNA fragments or with the two DNA fragments. The DNAH segments are highly similar. Later experiments (圔 11, 12) confirmed that the DNA sequence of RAMYC28-5 'does indeed have a high degree of homology with the DNA sequences of RAMYG28a and RAMYG6C. As for RAMYG28b and RAMYG6a, the weaker positive response may be due to the lower degree of homology between the DNA sequence of the probe and the two DHA segments. This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) -36-(Please read the precautions on the back before filling out this page). · · Βτ line. system

V. Explanation of the invention () B. Determine whether the known α "amylase cDN A may be derived from the gene group D Ν Δ that has been isolated by itself: using four α-amylase genes (RAMYC26, 27, 28, 30) The 3 'end is a molecularly specific probe for molecular hybridization to understand that the four cc-amylase cDNAs may be derived from the DNA of the gene group isolated from those ridges, and the hybridization results (Figure 3B (3D, 3F, 3G) shows that RAMYC26 is a pure cDNA strain derived from RAMYG8 'RAMYC27 and RAMYC28 are pure cDHA strains derived from RAMYG17 and RAMYG28, respectively (please refer to Table 1). 3. Rice cc-amylase gene The second transgenic group of DNA fragments was cloned from the pure DNA of the rice gene group DNA bank and the probes at the V-terminus and 3'-terminus of the coding region of the α-amylase gene were hybridized and reacted. (Figure 5), some of which were transfected into the pBlue.scr ipt HKS (+ / _) vector, as shown in Figure 6. Among them, RAMYG17a2 (Figure 4b) and RAMYG28a2 were cut by the restriction enzyme to cut down the DNA fragments, and then transfected into pBlue-script vector. Fourth, rice ex-starch water DN A sequence analysis at the 5 'end of the enzyme gene: (A) DNA sequence analysis at the 5' end of the RAMYG17 gene: The probe made from the 5 'end of the RAMYC30 coding region is hybridized with 1 ^ »¥ 01781 Reaction (Figure 2b), so RAMYG17ai may contain the α-amylase gene promoter site. Using restriction Sal I and EcoRI can reduce 4.5kb RAMYG17ai to 1.2kb RAMYG17a2 (Figure 4), while still being compatible with RAMYC30 The 5 'end reaction indicates that RAMYG17az may still contain the promoter site of the α-amylase gene, and RAMY was deleted by Εχο I (please read the precautions on the back before filling this page).

Q-Alignment · This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm)-37-402638 Printed by A7, Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ___ ^ _ ΒΠ ___ 5. Description of Invention () G. The DNA fragment of 17a 2 was gradually shortened, and then the DNA sequence analysis was performed by the Sanger double-deoxyribonucleic acid chain termination method. The completed DNA sequence was compared with the published rice alpha and starch hydrolase genes using the GCG system. . Although the DNA sequence of RAMYG 17 33 is only 1200 bp, it has been found to be identical to the λ 0Sg2 pure strain (Huang et al., 1990) (Figures 7 and 8). This means that RAMYG17 a2 and λ 0Sg2 pure strains should belong to Quoting a gene. According to published reports, the signal peptide site of the α-amylase gene can be found (as shown in Figure 8). There is an EeoN I restriction enzyme in the DNA sequence immediately after the site of the signal peptide chain The cleavable part, so use EeoNI to cut at the end, so you can cut out the 5 f end of different long gods containing different lengths of the ct-Dianfen hydrolase gene promoter and the DNA fragment containing the 3 'end peptide chain (Figure 9) In the future, these DNA fragments can be linked with genes to express proteins to construct a protein expression vector and secrete the expressed bamboo protein to the outside of the cell by signal peptide function. (B) DNA sequence analysis of the 5 'end of RAMYG6 and RAMYG28 genes: The probes made at the 5' end of the RAMYC28 coding region and RAMYG6 and RAMY CI28 all got positive hybridization reactions. The positive DNA fragments were named as RAMYG6a 'RAMYG6c, Ri \ MYG28a, and RAMYG28b (Figure 2F) After analysis of the DNA sequence, it was found that RAMYG28a and RAMYG6b both contain the 5' terminal promoter site of the α-amylase gene. The 5 'terminal promoter site and RAMYC28 code included in RAMYG6b There is no homology at the ends of the region. RAMYG6c ^ [J has, so the former does not hybridize positively with the probe, the latter has () 1 0). In the experiment, Huang et al. Published two 痼 oc-amylase genes (Please read the notes on the reverse side before filling out this page) © Threading This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) Printed by A7 B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Description of the invention () They named them RAmy3D and! IAmy3E ° After comparing the DNA sequences, 'RAMYG28 and RAmy3E were found to be the same pure strains (圔 10,11), while RAMYG6 is 3' across RAmy3E ' The site and almost the entire RAmy3D gene (圔 10, 11, 12). A study by Huang et al. (Huang et al., 1990) has shown that RAmy3D (= RAMYG6) and RAmy3E (= RAMYG28) are located on the same chromosome, two The distance between the subjects is about 4.0kb (_10), and this result is also confirmed by the DNA sequence analysis of the present invention. V. Structure of the GUS expression vector: A. Construct plastids pBSI, pBXI, and PBSM (Presented by Divid McElory) pBI 101.1, pBI 101.2 and pBI 101.3 (CLONTECH, No. 6022) were transfected with the promoter-deficient E. coli GUS gene (2kb) on the Agrobacterium tumifaciensbin ar y plastid vector p B LN 1 9 and the soil rod The terminator (260bP) of the nopaline synthetase (NOS) gene of the M Agrobacterium T i plastid is formed at the 3 'end of the GUS gene. Therefore, the promoter of the plant gene can be easily inserted in the GUS gene. The 5 'end of the polylinker is inserted, and the connected promoter is used to drive the transcription of the GUS gene, which can be used for research on gene regulation. Because pB IN 19 has a low replication efficiency in fine bells and is not easy to purify in large quantities, the restriction enzymes Bam HI and spel were used to terminate the 01 ^ gene and 1 ^ 0 contained in PBI101.1, pBI 101.2, and 98101.3, respectively. Zi Zi? The 81 ^ 19 carrier was isolated, and the DNA fragments of pBI 101.1 and pB 101.3 were inserted into the plastid PBluescritHKS (+) cut by the restriction enzymes Bam HI and spel to obtain plastids P B S I and p B S I, respectively. This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) -39-(Please read the precautions on the back before filling this page) Pack · 0 Orders © Printed by the Central Consumers Bureau of the Ministry of Economic Affairs A7 —__—: _ B7 _ 5. Description of the invention () The 01 ^ fragment of mouth 81101.2 is inserted to limit the enzyme 83111111 and the parental dagger 3 1 plastid 08! 1136 (31- 丨 01:] 11 ($ (+ ) To obtain plastid 98 <] 1 (_13 six). The difference between pBS I, pBX I, and pBS Iff is the polylinker site at the 5 'end of the GUS gene, and there is a nucleotide difference in each reading structure. The promoter can be inserted into a GUS expression vector by selecting a suitable reading structure. B. Transplant the GUS gene into a plastid with an alternative marker-hygromycin-resistant gene because PBSI, ρΒχΠ, pBSBI ( (Figure 13A) There are no markers to choose from, and it may be difficult to determine whether the gene has been successfully transplanted into plant cells when performing plant trait transformations. Therefore it is important to establish a plastid with co-selectable markers. Restrict PVU E to PBS I, pBX E and PBs The GUS gene in I was isolated from the pBluescript vector along with multiple implant sites T 7 and T 3 and the multiple implantation sites (Figure 13B), and then inserted into PTRA132 (_13〇) (835 ^ 3111111) cut with Hind M. 1; 067 31, 1990) vector, the results obtained? 8 father I-132 and pBSffi-132 (Figure 13D), the GUS gene transmission direction of this two plastids and the hygromycin resistance gene (Hph) transmission The direction of the GUS gene pBS 1-132 (Figure 1E) is opposite to that of the hygromycin-resistant gene, so a total of three plastids with GUS and hygromycin-resistant genes were obtained. All of them are 7.0 kb, and these three plastids can be used to analyze the functions of various gene promoters in the future. C. Construction of a fusion vector of rice α-amylase promoter and GUS gene This paper applies Chinese national standard (CNS) A4 Specifications (210X297 mm) -40-_______.% 1_ (Please read the notes on the back before filling out this page)-Order-Embroidery A7 B7 V. Description of the invention () 1. Construct a fusion vector of RAMYG17 promoter and GUS gene According to Figure 9, the restriction enzyme map and graphic illustration of R AMYG1 7a i have been obtained. pHE (0.88kb), PAE (1.7kb), PSE (2.4kb) and other three DNA fragments containing the promoter of the cf-amylase gene. These three DNAs were inserted into the plastid pBS cut by the restriction enzyme Sma I I (please refer to the description of the material method for the conjugation method). According to the principle of the gene triple code, decoding starts from the protein translation start point ATG of pHE, pAE, and pSE, until the translation start point ATG of the GUS gene must meet the triple code. Reading architecture. The completed promoter-GUS fusion vector is named pHE-I, pAE-I and pSE-I (please refer to 圔 14A, 14B, 140 ° printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the note on the back first) Please fill in this page for further details.) At this point, the laboratory has obtained plastid PTRA 132 (presented by Hayashimoto), and used the restriction enzyme pvull to convert the DNA of the promoter-GUS gene in pHE-1, pAE-I and pSE-1. The fragment, together with the T7 promoter and the T3 promoter and the multiple implantation sites therebetween, were isolated from the p β 1 uescri ρ t vector (Figure 14C), and then inserted into the pTRA 132 vector cut with Hindi (圔 14 D). The results were respectively Three fusion vectors with resistance to hygromycin were obtained, including pSE 132 (11.4 kb), pAE 132 (9.7 kb), and pHE 132 (7.9 kb). The translation direction and resistance of the GUS genes of pSE 132 and pAE 132 were obtained. Hygromycin gene translation direction is the same, but pHE 132 GUS gene translation direction is opposite to the hygromycin resistance gene translation direction.

2. Construct a fusion vector of RAMYG6, RAMYG28 promoter and GUS gene. The RAMYG6 and RAMYG28 gene promoters are located in the two DNA fragments of RAMYG6b and RAMY G28a2 (Figure 10). The α-starch of RAMY G6b can be hydrolyzed by EcoNI and Sal I DM sequence of the 5 'end of the enzyme gene and the signal peptide chain (the total paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm)) Λ 7402638 printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs ) About 1.6kb long) isolated from the plastid pBluescript. However, RAMYG28a2 cannot use restriction enzymes to isolate the DNA sequence of the 5 'end of the cc-amylase gene and the signal peptide chain * because there is no appropriate restriction enzyme available at the end of the DNA sequence of the signal victory. Therefore, the Exo M nuclease removal method was used to gradually shorten the DNA from the 3 'end of RAMYG28a2 until it was near the signal peptide DNA sequence (圔 11, at the thick arrow), and then hydrolyzed α-starch with the restriction enzymes APa I and EcoRV. The 5 'end of the enzyme gene and the DNA sequence of the signal peptide were isolated from the plastid pBluescr ipt. Therefore, according to the gene triple code principle, the two DNA fragments containing the promoter and signal peptide chain isolated from RAMYG6b and RAMYG28a2 were inserted to pBS 1-132, which was cut by the restriction enzyme H i nd M, to obtain 1 ^ 1, respectively. ^ 06-132 (8.61 < 1)) (圔 15) and 1 ^ ¥ 28 -132 (I) (8.2kb) (Figure 16) Fusion vector of α-amylase gene promoter and GUS gene . The gene 0 ¥ 06-132 and 1? Long ¥ 028-132 (1) the translation direction of the 01] 3 gene are the same as the translation direction of the hygromycin resistance gene. However, the signal sequence of the signal peptide chain and the GUS gene have a stop signal TGA (_ 15D, 16D). In order for the constructed fusion vector to ignore the signal of the α-amylase enzyme gene, the DNA sequence and the GUS gene will not generate a stop translation code, and the restriction enzymes Apal and EcoRV were isolated from the α-starch from RAMYG28aa. The 5 ′ end of the hydrolase gene and the DN A sequence of the signal peptide chain were inserted to pBSI-132, which was cut by the restriction enzyme clal, to obtain RAMYG28-132 U) (_ 17C). The translation direction and resistance to moisture of the GUS gene The translation direction of the mycin gene is reversed, and there is no termination between the DNA sequence of the signal chain and the GUS gene (please read the precautions on the back before filling this page). Standard (CNS) Α4 Specification (210X297 mm) -42-* — " I _____ ____ * — " I _____ ____ Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 402638 A7 B7 V. Description of the invention () Translation of the password Produces (圏 17D). 6. Transplant GUS expression vector into rice suspension culture cells by electro-penetration. By electroporation, three GUS expression vectors, including PHE132, PAE132 and RAMYG6-132, have been transfected into rice suspension culture cells. After 3 days, Xg 1 u was used to develop the reaction color, and the transient expression of the GUS expression vector was measured. The results showed that the transgenic cells of the three GUS expression vectors such as PHE 132, PAE132, and RAMYG6-132 had blue expression (Figure 18B, 18C). ), And the control group cells were transduced without the α-amylase gene promoter, and only the GDS gene-containing vector was used for transfection. After the same treatment, the cells did not appear colored (Figure 18A). This proves that pHE132, pAE 132 and RAMYG6-132 can all behave normally in rice cells. Whether PSE132, RAMYG28-132 (I), and RAMYG28-132 (I) can also perform normally in cells is to be confirmed by further experiments. M: Apply gene transfer technology to study the expression and function of eukaryotic genes to promote understanding of the regulatory mechanisms of higher organism genes. It is known that certain α-amylase genes in cells are subjected to glucose deficiency treatment in the medium of rice suspension culture cells, and a large number of α-amylase enzymes are also produced and secreted into the extracellular medium (Kuo, 1990). This phenomenon of a large number of ex-amylase genes induced by glucose deficiency, how the cell transmits the signal of glucose deficiency, how to sense the signal of glucose deficiency to regulate the large-scale expression of specific Ct-amylase enzymes, and -Starch, hydrolase is secreted outside the cell, which is the subject we are interested in studying. The case B was selected from the rice gene group D Ν Α bank to obtain 12 cabinets of different paper sizes that are applicable to China National Standard (CNS) Α4 specifications (2 丨 〇 father 297 mm). Factory: -------- ---- ο 装 —I (Please read the precautions on the back before filling this page), 1T. Line 402638 A7 _B7_ V. Description of the invention () (Please read the precautions on the back before completing this page) C (- Pure strains of amylase hydrolysate gene group DNA, using probe hybridization and DNA sequence analysis to further determine that at least 4 of the 12 alpha-amylase hydrolyzate gene group pure strains contain alpha-amylase gene 5 'end and DM sequence of signal peptide chain. The four pure strains are RAMYG6, RAMYG8, RAMY G17 and RAMYG28. Among them, it is worth noting that the probes made of RAMYG8 and RAMYC30 (Tzou, 1990) coding region. After hybridization, five DNA fragments showed a positive response (圔 1B). The total length of the five DNA fragments was about 10 kb. According to the DNA sequence analysis of RAMYG8C (Lo, personal communication), RAMYC 26 (Tzou, 1990) was derived RAMYG8 pure cDNA strain, and RAMYG8C contains the 5 'end of the RAMYC26 gene and the signal victory DNA sequence of the strand. 圔 3B shows that the specific gene probe made from the 3 'end of RAMYG8b and RAMYC26 shows a strong positive hybridization reaction. It is speculated that RAMYG8b contains at least the 3' end portion of RAMYC26 gene. Therefore, RAMYG8C and RAMYG8b contain The 5 'and 3' ends of the RAMYC26 gene. In addition to RAMYG8b and RAMYG8C, the three DNA fragments of RAMYG8a, RAMYG8d, and RAMYG8e have a total length of about 5.6 kb, so it is speculated that these three DNA fragments contain at least another 値 α-starch Hydrolysase genes, so RAMYG8 halo contains at least two alpha-starch hydrolase gene pure DNA strains. Table 1 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs shows RAMYG6 and RAmy3D * RAMYG17 and RAmylA, and RAMY G28 and RAmy3E is the same pure rice ct-amylase gene DNA strain, P0S137 (RAMYC6) is a pure cDNA strain derived from RAMYG6 'POS103 (RAMYC27) is a pure cDNA strain derived from RAMYG17, and a-Amy3 (RAMYC26) It is a pure cDNA strain derived from RAMYG8. RAmylA (RAMYG17) has higher DNA transcriptional activity than rice paper at the germination stage of rice seeds and roots and leaves, and is suitable for China Standard (CNS) A4 specification (210X297 mm) -44-402638 A7 B7 V. Description of the invention () Other known α-amylase genes of rice D (Huang eta 1., 1 990a). RAmy3 (RAMYG28) performed best in immature rice seeds (Huahg et al., 1990a) and when the medium was induced by glucose deficiency (Kuo, 1990). RAmy3D (RAM YG6) is most expressed in rice callus (Huang et al., 1990a). The similarities between RAMYG (RAiny3D) and RAMYG28 (RAmy3E) include: (a) the transcription direction of the two on the chromosome is the same * (b) both have 2 痼 interposers and the length of the interposers are similar, (c) The modified protein (Prepr oteei η) has similar numbers of amino acids, RAmy3D is 435 amino acids, RAaiy3E is 437 amino acids, (d) can be expressed in large amounts in callus (Huang et al. , 1990a). Although RAMYG6 and RAMYG28 have these similarities and are located on the same chromosome, the results of siot-blot method through DNA cross hybridization belong to different ethnic groups. RAMYG6 is ethnic group 2 and RAMYG28 is ethnic group 5 (Huang et al., 1990b ). Comparing the amino acid sequence of the signal peptide chain, RAMYG28 is similar to wheat Amy3 (Table 2), and RAMYG6 is significantly different from other cereal starch hydrolase genes. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page) The glycoproteins secreted by plants from outside the cell can be divided into two types according to their structure and function. The first category is proteins without enzyme activity. The oligosaccharides are combined with the hydroxyamidic acid of the peptide chain through a 0-glycosidic bond to form a non-enzymatic secreted protein (Fincher et al., 1983). The first type is a protein with enzyme activity. The oligo- and Asn-X-Thr / Ser in the peptide chain sequence (X represents other amino acids other than Asp or pro) are bound by N-sugar ridge bonds. Together form a secreted protein with enzyme activity (Akazawa et al., 1985) ° This paper size applies the Chinese National Standard (CNS) A4 specification (21 × 297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Carpling 402638 at B7 V. Description of the invention () The major difference between the α-spatter hydrolase of rice seeds and the α-starch hydrolase of other tadpole seeds is that there are many mannose and complex oligosaccharides that will bind to the mature polysaccharides of rice. Chain (M itsui & A kawaza, 198 6), but the mature a-amylase of barley and wheat is not glycosylated. In this case, RAMYG17 has a hypothetical Η-glycosylation site at positions 2450 and 3380 of the DNA sequence (Figure 8), and its amino acid sequences are Asn-Leu-thr * and Asn-g] y- Thr. Asn-Leu-Thr is located in the signal peptide chain. When the protein is secreted into the ER cavity, the signal peptide chain will be cut off. Therefore, the mature α-amylase produced by RAMYG17 is predicted to be only in Asn-gly -Thr is glycosylated by N-glycosylation. Kiiinagai et al. (1 990) _ Transplanted into yeast cells using POS103 (RAMYC27) and collected α-amylases secreted into the culture medium if deglycosylated drug TFMS (trifluoromethanesulfonic acid) Treatment of α-amylase, the molecular weight of α-amylase hydrolyzed by TFMS is smaller than that of oc-amylase not treated by TFMS, which proves that the mature cc-amylase produced by POS103 is indeed glycosylated . RAMYG6 has only a hypothetical N-glycosylation site at the signal peptide Asn-Thr-Ser (Figure 12), while RAMYG28 predicts that no place can be glycosylated (Figure 11). If the N-glycosyl group fc is used in accordance with the principle of Asn-X-Thr / Ser, the mature α-amylase produced by RAMYG6 and RAMYG28 will not be glycosylated (0 'Ne i U et al ., 1990). This is different from the conclusion that Miyata et al. (1982) believe that all rice α-amylases are glycosylated by °. This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297). (%) -46-(Please read the notes on the back before filling out this page) Order printed by the Central Consumer Bureau of the Ministry of Economic Affairs, printed by A7, B7, i. Description of invention () Table 2 lists rice, barley, and wheat. Comparison of Peptide Amino Acid Sequences. R AMYG 1 7 has two imaginary translation start points. The translation start point starts from the first methylmethionine, and its signal peptide chain has a length of 31 M amino acid. If the translation start point starts from the second When methionine starts, the signal peptide contains 25 amino acids. Von Heijne (1985) divided the signal peptide chain into three regions: η, c, and h. The η region is located at the N-terminus of the signal peptide chain and consists of small peptides with a positive electricity price of one or two. The c-region is located at the C-terminus of the signal peptide chain and is composed of approximately 5 amino acids. The h region is located between the η and c regions, and consists of about 7 amino acids to form a hydrophobic core of the signal peptide chain. Table 2 shows that barley and wheat have almost conserved amino acid sequences (S-L-S-S-G) in the c region of the signal peptide chain. Therefore, it is speculated that this sequence may be the identified position of the signal peptide chain (Kumagai et al., 1990). However, the c region of the signal peptide chain in rice does not have such a conservative amino acid sequence. Except for RAMYG28 with three positive electricity prices in the η region of the peptide chain of cx-amylase in Table 2, the rest have one or two positive electricity prices. In the h region of all signal peptide chains, there is a small segment of a hydrophobic core consisting of six or seven consecutive hydrophobic amino acids. This hydrophobic peptide may play a role of binding to the hydrophobic bilayer lipids of the endoplasmic membrane, and then guide the polypeptide chain into the inner cavity of the endoplasmic reticulum. It is also worth noting that the signal peptide chains of the α-amylases listed in Table 2 do not have acidic amino acids.

The three rice α-amylase promoters obtained by the present invention have been constructed as vectors for controlling the expression of GUS (Figures 14, 15, 16, 17), in which the RAMY paper size applies the Chinese National Standard (CNS) A4 specification ( 21〇X297 mm) -47 l · — — _____ 义 — 丨 (Please read the notes on the back before filling this page) Order printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 402638 Α7 Β7 V. Description of the invention ( ) 06-132 and 1? / ^ ¥ 028-132 (1), etc. 値 01] $ expression vectors at multiple implantation positions where the DNA sequence of the peptide bond of the amylolytic enzyme and the GUS gene are connected A stop translation signal TGA (D15D, 16D) is formed at the position 'Therefore, the N-terminus of the GUS protein precursor translated by these two GUS expression vectors should not contain the signal peptide of α-amylase, so GUS Nor can proteins be secreted outside the cell. The GUS expression vector was transfected into rice suspension culture cells by electro-penetration. It has been determined that pHE 132, pAE 132, and RAMYG6-132 can all express the GUS gene (圔 18), so it is proved that the PHE132 constructed in this experiment, Promoter-GUS gene fusion vectors such as pAE 132 and RAMYG6-132 can indeed perform normally in rice cells, and it is predicted that GUS proteins produced by PHE 132 and pAE 132 will be secreted outside the cell. Based on the earlier discovery by the inventors of the present invention that the lack of a carbon source in the medium can induce the expression of the α-amylase enzyme gene and the above experimental results, it can be inferred that the promoter of the ct-amylase gene and the signal peptide chain are used to guide protein secretion. Characteristics, can be engaged in the study of gene regulation and protein secretion mechanism, and then replace the GUS gene with other genes that are favorable to humans, under the control of the promoter of α-amylase gene in suspension culture in plants (such as rice) Cells are treated with carbon deficiencies, especially sugar deficiencies, to engage in mass production of engineered proteins and benefit the population. Although the present invention is described by the above-mentioned preferred embodiments, as can be understood by those skilled in the art, the present teaching can still produce a variety of changes, modifications and equivalent substitutions through the above teachings. The patent protection shall be limited by the attached definition of the scope of patent application and its equivalent replacement. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) _ 48-(Please read the precautions on the back before (Fill in this page)

402638 at B7 V. Description of the invention () Definition 0 Affirmed matters: A defined in the original items 13 and 14 of the scope of the patent application for this case contains A Amy6, α Amy7, and A Amy8, which are promoter regions of the ct-amylase gene Or a AmylO gene, which corresponds to the 12M pure strain obtained by the inventor of the present invention in the present invention (please refer to the description in the last line on page 43 to the line 18 on page 44 in the description of the case). The inventor originally used the laboratory's own naming symbols to identify the obtained pure plants, and then republished these pure plants by adopting the international naming method for publication of the paper. Among them: «△ 1 ^ 6 Corresponds to 1-4 ^ 1 ¥ 026 described in the description of this case; c (Amy7 corresponds to RAMYG17 described in the description of this case; 〇i Amy8 corresponds to RAMYG28 described in the description of this case; `` △ 1〇10 corresponds to ^ 1 ^ 030 described in the description of this case, and cc Amy3 corresponds to RAMYG6 ° described in the description of this case (please read the precautions on the back before filling this page). 0 Pack-Order Printed by a Consumer Cooperative of the Central Standards Bureau of the Ministry of Light Industry Applicable to China National Standard (CMS) A4 specification (210X297 mm) _ 49-Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 ___B7 V. Description of the invention () Formula description: Figure 1: Use RAMYC30 coding area as a probe The 14 strains of the genetic group obtained were screened to further analyze whether they were α-amylase enzyme genes. (A) (C): Pure DNA of the gene group selected from the rice gene bank to limit the enzyme Sa 1 I The DNA swimming picture after the autophagosome DN A was cut, separated by agar gel electrophoresis, and stained with ethidium bromide. RAMYG19 and RAMYG17 may be the same pure strain.

(B) (D) Southern blotting method was used to transfer the DNA on the agar colloid in (/ 〇 and (C) to the gene sieving membrane, and then hybridized with the probe made by the R AMYC30 coding region, and 4a was found. (9kb), 4b (2.3kb), 5a (6.5 kb), 5b (3.2kb), 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) Shows a hybridization reaction, so it is inferred that these DNA fragments may contain the cc-amylase gene. His several pure strains, such as RAMYG10, RAMYG11, RAMYG 14, RAMYG18, and RAMYG20, have no positive reaction, so they may not contain α-amylase Genes, or the α-amylase gene contained in them, have low DNA homology with the probe. The DNA fragments in Figures 1 (A) and (C) that react with the probe are marked with a circle, " "M" indicates the DNA marker of the lkb ladder. Figure 2: Identifying the 5 'end of the α-amylase gene: (A) (C) (E) (G): Using Sal I to automate the rice genome DNA Paper size applies to Chinese National Standard (CNS) Α4 size (210X 297 mm) J- --- 1 ----- II (Please read the precautions on the back before filling out this page) Order 638-Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Separated from DNA. Agar colloid Electrophoresis pictures after electrophoresis separation and eth 丨 di it mbr 〇mide staining, in which RAMYG18 and RAMYG28 may be the same pure strain. (B) (D) (F) (H): The 5 'end of the RAMYG28 and RAMYG30 coding regions are used. And the three probes made at the 3 'end (HK350) of HS 501. Through the results of molecular hybridization, RAMYG6, RAMYG8', RAMYG17, RAMYG18, RAMYG21, and RAMYG28 were shown to have a positive hybridization reaction, so it is highly likely that the five strains of pure strain s Promoter site containing α-amylase gene. The coding region of RAMYG18 and RAMYG30 in 圔 1 (D) does not show a positive hybridization reaction, but here it shows a positive hybridization reaction with the 5 'end of RAMYC28. The 5 'end and 3' end probes are both DNA probes prepared by in vitro methods. This probe contains a small plastid (ρΒ 1 -uescriptli KS (+ / —)) multiple implanted DNA Fragment, maybe this DNA and tag (lkb ladder, B The 1.6 kb and 0.5 kb in RL) have highly homologous DHA sequences, so they show a strong positive hybridization reaction at the positions marked with 1.6 kb and 0.5 kb. Figure 3: Screening of the oc-amylase gene group DNA and the three α-amylase cDNAs (RAMYC26, RAMYC27, RAMYC28, RAMY C30) at the 3 'end of the specific cause probe hybridization reaction, so that Confirm whether these cDNAs may be derived from the isolated gene group DNA 0 (A) (C) (E): Pure rice gene group DNA was cut with the restriction enzyme Sal I autophagosome DN A and subjected to agar gel electrophoresis Separate and ei th idi um This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) Binding. Order 'line-402638 at B7 V. Description of the invention ( ) Photograph of DNA electrophoresis after bromide staining. Those who respond to the D N A fragment and the probe in the picture are marked with a circle. (B) (D) (F) (G): Figures (A) (C) (E) X-ray film of hybridized DNA with specific gene probes for four specific genes of cDNA. The results show that RAMYG6, RAMYG18 and RAMYG28 may contain the genetic DNA of RAMYC 28, and RAMYG18 and RAMYG28 may be the same gene; RAMYG8 may contain the genetic DNA of RAMYC26; RAMYG21, RAMYG22, RAMYG23 may contain the genetic DNA of RAMYC27. In (F), RAMYG18 also reacts positively with the fixed gene probe at the 3 'end of RAMYC27. It is possible that the 3' ends of RAMYG18 and RAMYC27 also have DNA sequences with a similar composition to some extent, so they also exhibit positive hybridization reactions. Figure 4 (A) (B) The 5 'end of RAMYG17a (4.3kb) and RAMYC30 coding region has a positive hybridization reaction. Using double digestion of Sal I and EcoRI restriction enzymes, DNA can be reduced from 4.3kb to 1.2 kb while still having a hybridization reaction, thereby facilitating DNA sequence analysis. Printed by the Consumers' Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs I -------------- (Please read the precautions on the back before filling out this page) (C) (D) RAMYGISa (7.5 KB) and RAMYC28 The 5 'end of the coding region has a positive hybridization reaction. Using the dual digestion of two restriction enzymes, Sal I and EcoRV, the DNA can be reduced from 7.5kb to 1.8kb while still having a positive hybridization reaction. Sequence analysis. Figure 5: The results of the hybridization reactions in Figures 1, 2, and 3 are briefly summarized as shown in Figure 圔. The seven probes used in the experiment are all in different rectangular patterns. The paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) -52-Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Α7 Β7 5. Description of the invention (). The size of the rectangular pattern represents the strength of the positive hybridization reaction. The larger the pattern, the weaker the positive hybridization reaction. The dashed line in the box indicates that the same DNA fragment and two or three kinds of probes in the dashed line have a hybridization reaction. Figure 6 :

In Figures 1 and 2, a portion of the DNA fragment that is positive for hybridization with the coding of the α-amylase gene and the probe at the 5 'end site were cut to cut the DNA of the restriction enzyme Sa 1 I autophagosome and subjected to electrophoresis After the method was separated on the agar colloid, it was separated from the colloid [see Materials and Methods (1.2.3.1) for details], and then inserted into the pB 1 uescr * i pt to limit the incision. The size of these DNAH segments is in pBluescript. The size of these DNA fragments and the positions inserted in pBluescript are as expected (A) to (M). Figure 7: DNA sequence comparison of et al., 1990b). RAMYG17a and λ 0Sg2 are both 4.3 kb in length, but RAMYG17adU 5 'end is 1 lkb longer than λ 0Sg2 and 3' end is lkb shorter than λ 0Sg2. The DNA fragment (about 3.3kb) indicates that the DNA sequence of RAMYG17a and λ 0Sg2 are identical. Figure 8: Published DNA sequence analysis of A0sg2 (RAMYClA) (Huang et al., 1990b). Braces () (2100 ~ 3250) indicate RAMYG17a2, which is a DNA fragment whose DNA sequence has been determined by the inventors. The double line indicates the TATA box, the right arrow indicates the transcription initiation sequence, and the curve indicates that the 5 'end of the α-amylase gene of rice, barley, and wheat is covered by the guaranteed paper. The Chinese paper standard (CNS) Α4 specification ( 210 × 297 mm);-: ----- e equipment ------, order ------ ^ (Please read the precautions on the back before filling out this purchase) A7 B7 402638 V. Description of the invention () Obedient sequence.

The box line is a pyrimidine box, which is a conserved sequence contained at the 5 'end of many genes expressed by GA induction. The single-line signal indicates the amino acid sequence of the peptide chain, and the up arrow is the position of the DNA sequence cut by EcoN I. The positions of (1590) and (770) are Hind M and AccI. The dotted line indicates the amino acid sequence position that may be bound to calcium ion, and the thick line indicates the position of possible N-glycosylation. Figure 9: The 5 'end of RAMYG 17 contains promoters that can be cut to different lengths with restriction enzymes. The total length of RAMYG17 is about 12.6 kb, and the black solid area is RAMYG17a2, which is the completion of the DNA sequence analysis. 0} ^ sequence with the same 0 "fragment. Using 3311, RAMYGUaiU.Skb) can be separated from RAMYG17. The restriction map of RAMYG17ai is shown in the figure. Using Hindi! * AccI, Sail and EcoNI can cut RAMYG17ai into different contents. The DNA fragments of the long end and the signal peptide are called PHE (0.88kb), PAE (1.7kb), and PSE (3.4kb). B, E, S, X, and Xh in 圔 represent the restriction enzymes BamHI, respectively. , EcoRI, Sail, Xbal, Xhol. Figure 10: The relative positions and lengths of RAMYG6 and RAMYG28 and published RAmy3D, RAmy3E (Huang et al., 1990a). RAMYG28a and RAMY G6a are two adjacent α-starch hydrolysis Enzyme gene, 27bp in the middle was not transferred to the plastid. RAMYG6a and RAMYG6b may be mutually compatible. The paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) l ·!-—'--- --- ¾ — I (Please read the notes on the back before filling (This page) Order Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Printed by the Central Rubber Standards Bureau of the Ministry of Economic Affairs Printed by the Shelling Consumer Cooperatives of the Central Rubber Bureau 402638 A7-~ _B7 ____ 5. Description of the invention () The connection may also be separated, and enzymes are yet to be restricted It can only be known by map analysis. RAMYG6b and RAMYG6C are two connected DNA fragments. RAMYG6b and RAMYG28a2 are the parts of the DNA sequence that the inventor of this case has determined, and the length is 1.8 kb. RAMYG28a, RAMYG28a2, RAMYG6a and RAMYG6c are all Expressed in the same shape, the probes representing these DNA fragments and the 5 'end of the RAMYC28 coding region all show positive hybridization reactions. Figure 11: The leech ct-amylase gene RAmy3E (Huang et al., 1990) and RAMYG2 DNA sequence analysis of 8a3 The DNA in the braces (7 9 0-1 1 1 0) indicates the 5 positions of the coding region of the probe RAMYC28 used in Fig. 2. From 1320 (left arrow) to -630 The DNA sequence is the position of RAMYG28a2 (Figure 11). A negative sign indicates that the DNA sequence of RAMYG28a2W5 'is more than that of RAmy3E, and has about 630 nucleotides. The 3 'end (rightward arrow) from 1590 onwards is the same as the DNA sequence of RAMYG6a (see Figure 10). The double line represents TATA b ο X, and the curve represents rice, barley, wheat, -amylase groups. The 5 'end of the gene contains similar sequences. The horizontal line indicates the possible amino acid sequence of the peptide chain. There are a total of 25 amino acids. Dashed lines indicate the amino acid sequence sites that may bind to calcium ions. Figure 12: DNA sequence analysis of rice α-amylase gene "RAmy3E (Huang et al., 1990a) and RAMYG6b, RAMYG6c. The DNA in the braces (1350 ~ 2130) indicates that its DNA sequence is the same as that of RAMYG6C. The 5 'end of the DNA sequence is exactly the same as RAMYG6b. This paper size applies the Chinese National Standard (CMS) A4 specification (2 丨 〇 X 297 mm) one -55-(Please read the precautions on the back before filling this page )

402638 A7 B7 V. Description of the invention () RAMYG6b and RAMYG6C are connected with the recognition site of restriction enzyme Sail. The DNA (1380 ~ 1650) between the two thick arrows indicates a DNA sequence with high homology to the 5 'end of the coding region of the probe RAMYC28 used in the figure. The double lines indicate the TATA box, and the curves are the conserved sequences contained in the 5 'end of the ct-amylase gene of rice, barley, and wheat. A single line indicates the amino acid sequence of the possible signal peptide chain, and there are 2 amino acids in total. The dashed line indicates the possible amino acid sequence positions for calcium ion binding. The thick line represents the site of possible glycosylation. Figure 13: GUS gene transfected into plastids with alternative marker primary hygromycin resistance gene. (A) Plastid pBSI, ρβχΙ, pBSM map. At the 5 'end of the GUS gene, pB XE has one more nucleotide than pBS I, and pBS 2 [also has one more nucleotide than pBX II, which results in its reading structure having one nucleotide each. The difference, so that the promoter of the foreign gene and some genes can properly conjugate with the GUS gene and still conform to the principle of gene triple code. Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page) (B) (C) (D) (E): Use Pvu I to transfer the GUS gene and NOS terminator from pBSI, pBX I and pBS I were isolated (圔 B), and the 5 ′ and 3 ^ ends of this DNA fragment still contained multiple translocations of plastid PB 1 uescr · i pt. This DNA was inserted into the plastid PTRA132 (Figure C) cut with Hind M to obtain pBXE-132 and pBSH-132 (Figure D). The translation direction of the GUS gene was the same as that of the hygromycin resistance gene. The translation direction of the GUS gene of pBS I-132 (Figure E) is opposite to that of the hygromycin resistance gene. This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -56-402638 A7 __________B7__ V. Description of the invention () Figure 14: ct _ Lin powder hydrolase gene 5 'end contains a promoter site Transfer to plastids containing GUS and hygromycin resistance genes. (Please read the notes on the back before filling out this page) (A) (B) (C) (D) (E): Use restriction enzymes to cut out different promoter lengths pHE, pAE and pSE (Figure B) , And then inserted into plastid PBSI (圔 A) cut with Smal. As a result, three GUS expression vectors without pHE-I (5.95 kb), pAE-I (6.85 kb), and pSE-I (8.55 kb) (Fig. C) were obtained without alternative marks. Then the restriction enzyme pvlI was used to isolate promoters of different lengths together with GUS genes from f > HE-I, pHE-I and pSE-1, respectively, and then transferred to pTRAI 32, a plastid cut with the restriction enzyme Hind I (Figure D). As a result, three GUS expression vectors (圔 E) with hygromycin resistance genes were obtained. Among them, p A E13 2 and pSE 132 had the same GUS gene translation direction as the hygromycin resistance translation direction, and The translation direction of the GUS gene of PHE132 was opposite to that of the hygromycin resistance gene. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (F): DNA sequence analysis of the three junctions of PSE132, PAE132 and PHE132 with the 5 'end of the GUS gene. The first ATG is the translation initiation point of the α-starch hydrolase gene, and the second ATG is the translation initiation point of the GUS gene. The horizontal line indicates the DNA sequence of the expected signal peptide chain. Figure 15: The site containing the promoter at the 5 'end of the α-amylase gene is transferred to plastids containing GUS and hygromycin resistance genes. (A) (B) (C) The restriction sites EcoNI and Sail were used to isolate the promoter site of RAMYG6b (Figure 10) together with the signal peptide DN A sequence from pB 1 u -escript. Then insert the isolated promoter (Figure B) into the pBS M-132 (圔 A) vector cut with the restriction enzyme Hind M to obtain the Chinese paper standard (CNS) A4 size (2! 0X297 mm) for this paper size. ~-57-402638 A7 B7__ 5. Explanation of the invention () RAMYG6-132 (836kb) GUS expression vector (Figure C). RAMYG6-1 3 2 has the same translation direction of GUS gene. (D): DNA sequence analysis of the junction of RAMYG6-132 and the 5 'end of the GUS gene. The horizontal line indicates the predicted DNA sequence of the signal peptide. The nucleotide sequence TGA at the dotted line of the box represents the stop code. Figure 16: The site containing the promoter at the 5 'end of the α-amylase gene is transferred to a plastid containing dUS and anti-Hygromycin genes. (A) (B) (C) Using the restriction enzymes Apal and EcoRV, the promoter site of RAMYG28a2 (Figure 10) was separated from the peptid pBluescript with the DNA sequence of the signal peptide chain. Then insert the isolated promoter (Figure B) into the pBS I-132 (Figure A) vector cut with the restriction enzyme Hind II to obtain the GUS epitope vector of RAMYG6-132 (I) (8, 2kb) (Figure C ). The translation direction of RAMYG28-132's GUS gene is the same as that of the hygromycin resistance gene. (D): DNA sequence analysis of the junction between RAMYG28-132 and the 5 'end of the GUS gene. The horizontal line indicates the predicted DNA sequence of the signal peptide chain. The nucleotide sequence TGA at the dotted line of the box represents the stop code.圔 17: The site containing a promoter at the 5 'end of the amylase gene is transferred to a plastid containing GUS and a hygromycin resistance gene. (A) (B) (C): The restriction site Apal and EcoRV were used to isolate the promoter site of RAMYG28A2 (圔 10) together with the signal sequence of the peptide chain from the pBueue plastid. The isolated promoter (Figure B) was then inserted to pBS 1-132 (Figure 0.1.1 ^ 0282-1 32 (II)) cut by the restriction enzyme Clal and the translation direction of its GUS cells was similar to that of the hygromycin resistance gene. The paper size of the translated paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -58-__: -------- Q Pack— _ (Please read the notes on the back before filling this page) Set the central standard of the Ministry of Economic Affairs Printed by the Bureau ’s Consumer Cooperatives 402638 Printed by the Central Standards Bureau of the Ministry of Economic Affairs ’s Consumer Cooperatives A7 B7 V. Description of the invention () Direction is opposite. (D): RAMYG28-132 U) DNA that is connected to the 5 'end of the GUSGUS gene Sequence analysis. The horizontal line indicates the predicted DNA sequence of the peptide chain. Figure 18: GUS expression vector was transfected into rice suspension culture cells by electro-penetration, and GUS analysis was performed after 3 days of culture. (A) Cells in the control group were transfected with a promoter containing no α-amylase gene and a vector containing only the GUS gene. After 3 days of culture, they reacted with X-g 1 u and no basket color appeared. (B) The cells transfected with the GUS expression vector were stained with 乂 -§111 after 3 days of culture. The blue ones in the picture indicate the cell clusters that can be represented by 卩 1] $. (C) An enlarged photo of colored cell pellets after Xg 1 u reaction. The paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -59-(read first, read the first (Please fill in this page for matters needing attention), vs I TK-.

402638 A7 B7 V. Description of the invention (Table 1: Naming of rice α-amylase gene and its performance in cultured rice cells. The table lists the laboratory and the laboratory owned by Rodr · guez in the United States. Pure rice α-amylase gene DNA and pure cDNA strains. According to this table, RAMYG6 and RAmy3D, RAMYG17 and RAmylA, RAMYG28 and RAmy3E are the same α-amylase gene DNA pure strains, and RAMYC6 and P0S137, RAMYC27 and P0S103, RAMYC26-RAMYC27, RAMYC 28 and other pure α-amylase hydrolase cDNA strains are derived from RAMYG6, RAMYG8, 1.8 to 11 and 17 and 1 ^! ^ 028, etc. ○ £ -amylase gene group In rice callus, RAmy3D (RAMYG6) showed the strongest performance, and RAmy3E (RAMYG28) showed the strongest performance in the medium of rice suspension culture cells treated with glucose deficiency. 'Table 1.

Rodriguezs Lab «| ^ Within the performance _ ||; lack of wide; __ 一 ----- ύ costume—I (please read the precautions on the back before filling this page) Ordered by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economy system

Tu's Lab genome's cDNA genome RAMYG6 RAMYC RAmy3Db RAMYGfi (RAMYG26) RAMYC2 6r RAMYG17 (RAMYG27) RAi0nt27r RAmy 1AC (X0Sg2) RAMYG26 RAMYC25r RAmy3Eb RAMYC3〇y cDNA pOS137 (*-λ Ή * ⑺ Line * Department of Genetics, University of California, Davis, CA 956 16 # USA bHuang etal. # 199〇a cHuang etal., L99〇bd〇NeUl etal., 199〇 ^ GenBanic Data XHwa, i99 1 'yTzou 199〇ic Kuo, 199〇 This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -6〇- 5. Description of the invention (402638 A7 B7 Table 2: Signals of α-amylase from grasses A comparison of the amino acid sequence of the tritium chain. The thin horizontal line indicates the conserved sequence in the C-terminal region of the monopeptide chain amino acid sequence of the cereal α-amylase. However, the signal peptide chain of rice does not seem to have This conserved sequence. The thick horizontal line indicates the hydrophobic core composed of about 6 to 7 amino acids in the signal peptide chain. 'In the N-terminal region of the signal peptide chain, except for RAMYG28, which has three positive electricity valences Except the rest There are one or two positive electricity prices. Table · (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economy Printed species isomers Pure rice unknown | RAMYG17 Rice unknown Unknown Amy3 Barley X i7 High Pi pHV19 Barley_ High pi pM / C Barley High pi Amy64 Barley High Pi Amy46 Barley 髙 pi 1-28 Wheat. 髙 pi Amy 1 t Wheat High pi Amy 1-13 Barley Low pi CloneE Barley Low Pi Amy32 / B wheat low pi Amy2 / 54 amino acid sequence

liQVLNTnVNKHFLSLSVL IVLLGLSSNLTAG hKNTSSLCLL.LLVVLCSLTCNSGGA +++ _ _ MGKHHVTLCCVVFAVLCLASS LAQA hGKHSATLCGLLWVL CLASS LAQ A hANKHtSLSL.FLVLLGLSASLASG hANKHLSLSL.FLVLLGLSASLASG MANKHLSLSL.FLVLLGLSASLASG MANKHMSKSLF1V.LLGLSCSLASG hANKHLSLSL.FLVLIGLSAGLASG. MANKHLSLSL.FLVLLGLSASLASG- MASLHLSLS .FLVLLGLSASLA5J5. nGKNGSLCCFSLLLLLLLAGLL-ASG MG ^ NGNL'CCFSLLLLLL AGL ASG MGKNGNLCCFSLLLLLL GF ASG

This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) -61 402638 A7 B7 V. Description of the invention () MS Peiji base 0 stock solution. NH4N〇3 62.5 g / 1L 4 3 (5〇X ) KN〇3 95 g / 1L Caci2.H20 22 g / 1L (5〇X) Ξ MgS04.7H20 16.5 g / 1L MnS04 * H20 O.M5g / 1L (5〇X) ZnS〇4.7 7H2〇0.425 g / ^ MS Peyto '(looo mi) No. 1 --- 5 20 m] No. 6 1 ml. TPJi. 10 ml inositol. IOml (lOg / 1L) 2.4 -D 10 ml (10 uM) sucrose- 3〇g ρΗ ^ 5.δ 0 KH2P〇4 h3bo3. ΚΙ Na2M0〇4 * 2H2〇6.5 g / 1L 0.31 g / 1L 4 1.5 rng / 1L 12.5 mg / 1L (50〇) Na2-HDTA 14.65 g / 1L (5〇X) Fe2S〇4. 7H2〇1.39 mg / 1L, CuS〇4 * 5H2〇25mg /; L (1000 X) CoC12. 6H2〇25 mg / 1L 0 T.PJi .: Sembcorp-HC1 lg / Π- Vitamin Bir-HCl lg / 1L (100 X) Niacin +; 10g / a. (Please read the precautions on the back before filling this page)

1T # I. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs. Appendix 1. The paper size is applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm) A7B7 5. Description of the invention () Washing medium CPW7.4 1. CPW A: 5〇X / 1000 ml KH2P04 1.36 g KN〇3 O.505 g MgS04-7H20 12.3 g KI 0.006 g cuso4 * 5H2o 0.00125 g plus H20 to 1000 ml 2.CPWB: -50〇 / 1000 ml Cacl2. 2H20 74 g plus: H20. To 1000 ml * 3- CPW 7.4 CPW A: 2 cc CPWB 2 cc Ganzitol: 7.29 g force H20 to 100 mi and adjust · 'PH g 5.d with germanium (please (Please read the notes on the back before filling this page.) Description of the invention () A7 B7 Petri culture medium: KPR m: * 50 X / 100 ml 5. TPN: 100 X / 100 ml NH4N〇3 3 g nicotinic acid 10 mg KN〇3 9.5 1 g vitamins • 10 mg MgS04 * 7H20 1.5 g vitamins * 100 mg KH2 PO4 0.85 g in h2 0 to too ml Kcl 1.5 a „y 6. KPR ( IX): .in * h2o M too ml. * KM 2 ml B5 2 ml B5: 50 X / 100 ml MS2 2.0 mlNa2ii〇04 * H20 12.5 mg MS5 2 ml K! 3.75 mg TPN! Ml H3BO3 15 mg muscle Alcohol tml (tg / 100ml) 2.4-DI ml (22mg / 100ml) MnS04 · H2 〇50 mg NAA j ml (6.6mg / 100ml) ZnS04- 7H20 10 mg kallikrein 0.25 ml (21.5mg / i〇〇ml) ) CuS04_ 5H20 0.125 mg to g CoCl2 * 6H20 0.Ϊ25 mg .E sugar '0.025 g Add: H20 to 100 ml Add H20 to 100 ml and: M pH to 5.8 MS2 50 X / 100 ml Cacl2- 2H20 * 2.2 g plus H2O to 100 ml MS5: 50 X / 100 ml Fe504 * 7H20 0.139 g Na2EDTA 1.863 g plus H20 to 100 ml Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs ^ Please read the first three (Please fill in this page again)

This paper size applies to China National Standard (CNS) A4 (210X 297 mm) -64-References

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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. A7 B7 402638 i'invented Explanation () _1 Summary of Xixi (please read the notes on the back before filling out this page) _ 1: Pure strain of 14 値 1 gene group screened by using the RAMYC30 coding region as the probe, of which U), (C) DNA electrophoresis photos of DNA agar gel electrophoresis separated and ethidium bromide stained. (B), (D) Southern blotting method of DNA on agar colloid of (A), (C). The circle mark indicates DHA. The fragment reacts with the probe '"M" represents a DN A label of 1 kb 1 adder. Figure 2: Determining the 5 'end of the a-amylase gene: (A) (C) (E) (G): Sail was used to isolate rice genomic DNA from autophagosome DNA and separated by agar colloid electrophoresis and ethidium br 〇mide staining electrophoresis pictures; (B) (D) (F) (Η): the use of RAMYG28 and RAMYG30 coding region of the 5, and 3 'end of HS 501 (HK350) molecules of three probes Hybridization results.圔 3: Hybridization result of the selected α-amylase gene group DNA and specific gene probes made at the 3 ′ end of four α-amylase enzymes eDNAUAMYC26, RAMYC27, RAMYC28, and RAMY C30). Printed by the Consumer Bureau of Standard Bureau (A) (C) (E): The pure DNA of rice gene group was cut through the restriction enzyme Sal I autophagosome, and the DNA was separated by Qian Qing lipid colloid electrophoresis and eithidium bromide staining. Photo, the circle indicates that the DNA fragment reacts with the probe; (B) (D) (F) (G): _ (A) (C) (E) DNA and specific genes of four specific cDNAs X-ray negative image after genetic probe hybridization. Figure 4: This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 402638 A7 B7; .14 ^ ΙΜΙ5 · Μ ° * 隹 ΙΠΓ Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs () (A), (B) shows that the 5 'end of RAMYG17a (4.3kb) and RAMYC30 coding region has a positive hybridization reaction, using the dual digestion of sa 1 I and E c 〇RI restriction enzymes; (C), (D) shows the hybridization reaction of the 5 'end of the RAMYG18a (7.5kb) and RAMYC28 coding region using the dual digestion of two restriction enzymes, Sa II and EcoR V. Figure 5: A simple summary is derived from the results of the hybridization reactions in Figures 1, 2, and 3. The seven probes used in the experiments are represented by different rectangular patterns, and the size of the rectangular pattern 傜 represents the strength of the positive hybridization reaction. 》 The dotted line in the box indicates that the same DNAK segment and two or three kinds of probes in the dotted line have a hybridization reaction. Figure 6: The part shown in Figures 1 and 2 hybridizes with the ct-amylase gene and the probe at the 5 'end of the DNAH segment and is inserted into pBluescript, a plastid cleaved by the restriction enzyme Sail. And the size of these DNA fragments and the positions inserted in pBluescript are as shown in (A) to (M). Figure 7: Comparison of the DNA sequence of RAMYG17ai and A0Sg2 (Huang et al., 1990b), where the DNA fragment (about 3.3 kb) within the dashed line indicates that the 1 "0 \ 〇173 and the 0" sequence of human 0562 are identical. Figure 8 : Published A0sg2 (RAMYClA) (Huang et al., 1990b) (Please read the precautions on the back before filling this tile).

、 TT 'd This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) -73-402638 A7 —___ Β7 _ 5. DNA sequence analysis of the description of the invention (), where the braces () (2100 ~ 3250) Table 7rcRAMYG17a2, double line table τγϊΤΑΤΑ box. The right arrow indicates the sequence of the transcription initiation site. The curve indicates the conserved sequence contained in the 5 'end of the cx-amylase gene of rice, barley, and wheat. P yrimidineb ο X, a single line indicates the amino acid sequence of the signal chain, the up arrow is the position of the DNA sequence cut by EcoNI, and the positions of Hindi and Ac cl are shown at (1590) and (770), and the dotted line indicates The amino acid sequence positions that may be bound to calcium ions are shown. The thick line indicates the possible N-glycosylation position. Figure 9: The 5 'end of R AMYG17 contains a promoter that can be cut with different lengths by restriction enzymes. (The total length is about 12.6kb), where the black solid area is RAMYG17a2, the limit spectrum of RAMYG17ai is shown in the figure, and Hindi !, AccI, Sai, and EcoNI refer to RAMYG17ad; D into PHE (0.88k b), PAE (1.7kb), PSE (3.4kb) fragments, B, E, S, X, Xh represent the restriction enzymes BamHI, EcoRI, Sail, Xbal, XhoI ° Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the notes on the back before filling this page) Figure 10: The relative positions and lengths of RAMYG6 and RAMYG28 and published RAmy3D and RAmy3E (Huang et al., 1990a), of which RAMYG28a and RAMYG6a are two adjacent ones α-amylase gene, RAMY G6b and Ri \ MYG6 (^,! j are connected DNA fragments; RAMY G28a, RAMYG28a2, RAMYG6a, and RAMYG6c are all in the same figure. This paper scale applies Chinese national standard (CNS ) A4 specification (210X297 mm) 402638 i. Description of invention (). Figure 11: Rice α-ίdian powder hydrolase gene RA my 3 E (Huangeta I., 199 0) and RAMYG28a2te DNA sequence analysis, of which The DNA in brackets (790-1110) indicates the 5 positions of the coding region of the probe RAMYC28 used in Figure 2. The DNA sequence from 1320 (left arrow) to -630 is the position of RAMYG28ad! L, negative Number indicates that the 5 'end of RAMYG28a2 has more DNA sequences than RAmy3E The 3 'end (rightward arrow) from 1590 is the same as the DNA sequence of KAMYG6a. The double line indicates the TATA box, and the curve represents the 5' end of the α-amylase gene of rice, barley, and wheat. For similar sequences, the horizontal line indicates the possible amino acid sequence of the peptide chain. There are 25 amino acids in total, and the dashed line indicates the amino acid sequence site that may bind to calcium ion. Figure 12: Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Rice α-ί dianfan hydrolase gene "RA my 3 E (Huangeta 1., 1990a) and RAMYG6b Analysis of the DMA sequence of RAMYG6c. The DNA in the braces (1350 ~ 2130) indicates that its DNA sequence is the same as that of RAMYG6c. From 5 to 1350, its DNA sequence is exactly the same as RAMYG6b. The DNA (1380 ~ 1650) between the two thick arrows indicates the DMA sequence with high homology to the 5 'end of the coding region of the probe RAMYC2S used in Figure 2. The double line indicates the TATA box, and the curve is rice, 5 'of the ct-amylase gene of barley and wheat This paper size is applicable to China National Standard (CNS) 8-4 specifications (210X; 297 mm) _ 75-Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 i 、 Explanation of the invention () Conserved sequences contained in the terminal parts. The single line indicates the possible amino acid sequence of the tritium chain. The dashed line indicates the possible calcium ion-bound amino acid sequence position. The thick line indicates the possible sequence. Figure 13: GUS gene is transfected into plastids with alternative marker primary hygromycin resistance genes, where (A): maps of plastid pBSI, pBXE, pBSM; (B ): Pvu I was used to separate the GUS gene and NOS terminator from pBSI, pBX I, and pBS II; (C): Inserted this DNA into plastid PTRA132 cut with Hind M; (D): pBXE-132 and pBSM-132 (圔 D), the translation direction of the GUS gene is the same as that of the hygromycin resistance. 0 (E): The translation direction of the GUS gene of pBS I-132 is opposite to the translation direction of the hygromycin resistance gene. Figure 14: The site containing the promoter at the 5 'end of the α-amylase gene is transferred to a plastid containing GUS and hygromycin resistance genes, where (A) (B) (C) (D) (E): Using restriction enzymes, RAMYG17 was cut out with different promoter lengths pHE, pAE and pSE (Figure B), and then inserted into pBS I (Figure A), which was cut with Sma I. As a result, ρΗE- I (5.95kb) was obtained. pAE-I (6.85kb) and pSE-I (8.55kb) (Figure C) are three g US expression carriers without optional marks. The Chinese paper standard (CNS) A4 is applicable to the paper size. 21〇 × 2.97 mm) (锖 Read the notes on the back and note 戽 Fill this page first] Order 40S638 A7 ________ B7 _ ^ __ 5. Description of the invention () Use the restriction enzyme pvu I to combine promoters of different lengths with GUS Genes were isolated from pHE-I, pHE-I, and pSE-I, respectively, and then transfected into plastid PTRA132 (Figure D) cut with restriction enzyme H ind 11. As a result, three genes with hygromycin resistance were obtained. GUS expression vector (Figure E); (F): DNA sequence analysis of the three junctions of pSEi32, PAE132 and PHE132 with the 5 'end of the GUS gene. The first ATG is the translation start point of the ct-amylase gene, the second ATG is the translation start point of the GUS gene, and the horizontal line represents the expected DNA sequence of the peptide chain. Figure 15: The promoter containing the 5 'end of the α-amylase gene printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs was transferred to a plastid containing GUS and hygromycin resistance genes, of which (A) (B) (C) The restriction site EcoNI and Sail were used to isolate the promoter site of RAMYG6b (圔 10) together with the signal sequence of the peptide chain from pBlu-escript. Then insert the isolated promoter (Figure B) into the pBS I-132 (圔 A) vector cut with the restriction enzyme Hind ® to obtain the GUS expression vector of RAMYG6-132 (836kb) (Figure C); (D) · DNA sequence analysis at the junction of RAMYG6-132 and the 5 'end of the GUS gene. The horizontal line indicates the predicted DNA sequence of the peptide chain, and the nucleotide sequence TGA at the dotted line of the box represents the stop code. Drawing 16: The promoter-containing site at the 5 'end of the α-amylase gene is transferred to a plastid containing GUS and anti-Hygromycin genes, where ----- (Please read the precautions on the back before filling this page) This Paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) _ 77-402638 A7 B7 V. Description of the invention (.) (A) (B) (C) Use restriction enzymes Apal and EcoRV to convert RAMYG28a2 (圔 10) The promoter site and the DNA sequence of the signal peptide were isolated from the pBlnescript plastid, and the isolated promoter (圔 B) was inserted into the pBSI-132 (Figure A) vector cut by the restriction enzyme HindJI to obtain RAMYG6- 132 (I) (8.2kb) GUS expression vector (Figure C); (D): DNA sequence analysis of RAMYG28-132 and the 5 'end of the GUS gene, the horizontal line represents the predicted signal peptide DNA Sequence, the nucleotide sequence TGA at the dotted line of the box represents the stop codon. Figure 17: The site containing the promoter at the 5 'end of the α-amylase gene is transferred to a plastid containing GUS and hygromycin resistance genes, where (A) (B) (C): the restriction enzymes Apal and EcoRV are used The promoter site of RAMYG28A2 (Figure 10) was isolated from the plastid PB 1 uescr i pt together with the DNA sequence of the signal peptide, and the isolated promoter (Figure B) was inserted to pBSI-132, which was cut by the restriction enzyme Clal. (Figure C); (D): RAMYG28-132 U) Analysis of the DNA sequence at the junction of the 5 'end of the GUSGUS gene. The horizontal line represents the predicted DNA sequence of the signal peptide. Figure 18: GUS expression vector was transfected into rice suspension culture cells by electro-penetration, and GUS analysis was performed after 3 days of culture. U) Cells in the control group were transfected with a promoter containing no α-amylase gene and a vector containing only the GUS gene. After 3 days of culture, X-glu was used. This paper is in accordance with Chinese National Standard (CNS) A4 specifications (210 X 297 mm) -78-(Please read the notes on the back before filling out this page) Order M · Printed by the Consumers Cooperative of the Central Standards Bureau, Ministry of Economic Affairs, printed A7 B7 V. Invention Description () The response did not appear in blue. (B) Cells transfected with the GUS expression vector are colored with X-glu after 3 days of culture. Those with basket color in the figure indicate cell clusters that the GUS gene can express. (C) Enlarged photo of blue cell pellets colored by the xg 1 U reaction (Please read the notes on the back before filling in this page. (CNS) A4 size (210 X 297 mm) -79-

Claims (1)

1. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 6. Application for Patent Reexamination Case No. 86101436. Request for a revision of the patent scope. Date of revision: May 89. A method for a vector, which includes integrating a DNA sequence encoding a promoter and a signal peptide bond of an alpha tender hydrolase gene derived from angiosperms into a vector to regulate the structure of the vector encoding a desired protein The expression of the gene, so in the absence of sugar or lack of sugar, the promoter of the α-amylase gene derived from the angiosperm can induce the expression of the gene encoding the desired protein, and the βα derived from the α of the angiosperm -The peptide chain of the amylolytic enzyme gene can facilitate the secretion of the desired protein to the outside of the host cell so as to recover the desired protein from the culture medium, wherein the promoter encoding the powdery hydrolase gene and the DNA sequence of the signal peptide chain Lines are derived from angiosperms other than rice. 2. The method according to item 1 of the patent claim, wherein the sequence encodes a promoter and a signal peptide bond of a monocotyledon α-amylase enzyme gene 3. The method according to item 2 of the patent application, wherein the DNA The sequence encodes the promoter and signal peptide bond of the a_amylase gene of the grass family. 4. The method of claim 3, wherein the grass family is barley, wheat, rye or corn. 5. The method according to item 丨 of the patent application range, wherein the induction of the expression of the gene encoding the desired egg is a condition of lack of sugar in the medium. It is particularly preferable to lack sucrose, glucose, and fructose. The paper size applies to the Chinese national standard (CNS > A4 specification (210 x 297 mm white matter) (please read the note on the back? Matters before filling out this page) 1. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 6. Application for Patent Reexamination Case No. 86101436. Request for a revision of the patent scope. Date of revision: May 89. A method for a vector, which includes integrating a DNA sequence encoding a promoter and a signal peptide bond of an alpha tender hydrolase gene derived from angiosperms into a vector to regulate the structure of the vector encoding a desired protein The expression of the gene, so in the absence of sugar or lack of sugar, the promoter of the α-amylase gene derived from the angiosperm can induce the expression of the gene encoding the desired protein, and the βα derived from the α of the angiosperm -The peptide chain of the amylolytic enzyme gene can facilitate the secretion of the desired protein to the outside of the host cell so as to recover the desired protein from the culture medium, wherein the promoter encoding the powdery hydrolase gene and the DNA sequence of the signal peptide chain Lines are derived from angiosperms other than rice. 2. The method according to item 1 of the patent claim, wherein the sequence encodes a promoter and a signal peptide bond of a monocotyledon α-amylase enzyme gene 3. The method according to item 2 of the patent application, wherein the DNA The sequence encodes the promoter and signal peptide bond of the a_amylase gene of the grass family. 4. The method of claim 3, wherein the grass family is barley, wheat, rye or corn. 5. The method according to item 丨 of the patent application range, wherein the induction of the expression of the gene encoding the desired egg is a condition of lack of sugar in the medium. It is particularly preferable to lack sucrose, glucose, and fructose. The paper size applies to the Chinese national standard (CNS > A4 specification (210 x 297 mm white matter) (please read the note on the back? Matters before filling out this page) 6. The scope of the patent application A8 B8 C8 D8 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 6. If the patent & method is declared, which is conducive to the selection of angiosperm host cells transposed by the vector, The vector further contains a marker gene, a t ± k guide gene, an antibiotic-resistance gene, an enhancer, or a regulatory sequence as a selection marker. The method of patent claim 6 of the patent claims wherein the vector further contains an antibiotic-resistance gene as a selection marker. 8. If the scope of patent application is the 7th ^ method of coin shell, where the antibiotic targeted by the antibiotic-resistance group is kanamycin (hypermycin) or hygromycin (°) The method of item 6, wherein the vector further contains a reporter gene as a selection marker. 10. The method according to item 9 of the scope of patent application for β month, wherein the reporter gene is a glucuronidase gene. 11. Such as the scope of patent application! The method of clause, wherein the desired protein is / 5-glucuronidase. 12. A method for obtaining a desired protein by expressing a gene encoding a desired protein in an angiosperm cell, comprising: a) constructing a vector capable of expressing a gene in an angiosperm cell, the vector including a derivative From the angiosperm α-amylase gene promoter region and a gene encoding a desired protein, the DNA sequence of the promoter encoding the α-amylase gene is derived from angiosperms other than rice; b) with This vector transforms a proper angiosperm host cell; c) cultivates the trait-transformed host cell; it does so (please read the precautions on the back before filling out this page) • 2-six patent scope d) The cultivated trait-transformed host cell introduces a sugar-deficient or sugar-free condition to promote the expression of the gene under the control of the promoter region; and e) recovers a gene product expressed by the gene. 13. The method according to item 12 of the scope of patent application, wherein the DNA sequence encodes a promoter and a signal peptide bond of a monocotyledon alpha-amylase gene. 14. The method according to item 13 of the patent application, wherein the DNA sequence encodes the promoter and signal chain of the alpha-amylase gene of the gramineous plant. 15. The method according to item 14 of the patent application, wherein the grass family is barley, wheat, rye or corn. 16. The method of claim 1, 2, 13, 4, or 5, wherein the promoter region derived from the α-amylase enzyme gene includes a _amylase enzyme promoter and an alpha-starch The DN A sequence of the hydrolase signal peptide chain. 17. The method according to item 2 of the patent application, wherein the gene product is recovered from the cell culture medium of the trait-transformed host cell. 18. The method of claim 12 in which the vector is transferred to the host cell by a regulated trait transformation system, a polyethylene glycol (PEG) -regulated trait transformation, a poly-L ornithine method , Carrier acid method, microinjection method, particle impact method, electrical penetration method and ultrasonic method. 19 · Method for applying item 18 in the scope of patent application 'wherein the vector to the host -3- 297 mm) A8 B8 ----__ C8 Mountain ------------- __ VI. Application scope ~ ~ --— The transfer of cells is through a trait-regulating system such as 〇6. The method of claim 12 in which the appropriate primary angiosperm cell line is a suspension culture cell of a grass family plant. I The method according to item 2G of the Shenqing patent, wherein the appropriate angiosperm host cell line is rice, barley or wheat suspension culture cells. ★ The method of claim 21, wherein the appropriate angiosperm host cell is a rice suspension culture cell. 23. The method according to item 12 of the scope of patent application, wherein the sugar-deficiency or sugar-free condition is a condition of sucrose, glucose or fructose deficiency. 24. The method of claim 12 wherein the gene product is a protein of animal, plant or microbial origin. 25. The method according to item 12 of the patent application, wherein in order to facilitate the selection of angiosperm host cells transfected by the vector, the vector further contains an identification gene, a reporter gene, an antibiotic resistance gene, an enhancer or The sequence is regulated as a selection marker. 26. The method of claim 25, wherein the vector contains an antibiotic-resistance gene as a selection marker. 27. For the method of applying for the item% of the patent scope, wherein the antibiotic targeted by the antibiotic_resistance gene is kanamycin or hygromycin ° 28. For the application of the patent scope item 25 The method, wherein the vector further contains a reporter gene as a selection marker. 29. If the method of applying for the scope of patent No. 28, the reported gene is cold _ (Please read the notes on the mouth before filling in this page) A8 B8 C8 D8 6. Scope of patent application Glucononidase (/ 5-glucuronidase) gene. 30. The method according to claim 12 of the patent application, wherein the desired protein is glucoglucosidase. A method for obtaining a desired protein by expressing a gene encoding a desired protein in an angiosperm cell, comprising: a) constructing a vector capable of expressing a gene in an angiosperm cell, the vector including a derivative The promoter region of the α-amylase enzyme gene from angiosperms and a gene encoding a desired protein, the promoter region derived from the 0: -amylase enzyme gene includes the promoter of the α-j enzyme hydrolase gene And-the DNA sequence encoding the signal peptide chain of the α-amylase; the DNA sequence encoding the promoter and signal peptide chain of the α-amylase is derived from angiosperms other than rice; b) the The carrier trait is transformed into an appropriate angiosperm host cell; c) the trait-transformed host cell formed by culturing in a cell culture medium; d) the cultivated trait-transformed host cell is brought to a sugar-deficiency or sugar-free state to promote The expression of the gene under the control of the promoter region; and e) recovering the gene product expressed by the gene from the medium. 32. The method of claim 3, wherein the DNA sequence encodes a promoter and a signal peptide bond of a monocotyledon alpha-amylase gene. 33. The method according to item 32 of the scope of patent application, wherein the DNA sequence is edited on a scale of ㈣㈣297 6. Scope of patent application Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, the promoter and signal peptide bond of the alpha-amylase gene of the grass family plant. 34. The method according to claim 33, wherein the grass family is barley, wheat, rye or corn. 35. The method of claim 31 in the scope of patent application, wherein the transfer of the vector to the host cell is through a regulated trait transformation system, a polyethylene glycol (PEG) -regulated trait transformation, poly-L ornithine Method, calcium filling method, microinjection method, particle impact method, electro-penetration method and ultrasonic method. 36. The method as claimed in claim 35, wherein the transfer of the vector to the host cell is by a trait transformation system. 37. The method as claimed in claim 3, wherein the appropriate angiosperm The host cell line is a suspension culture cell of a poaceae plant. 38. The method of claim 37, wherein the appropriate plant host cell is a suspension culture cell of rice, barley or wheat. 39. The method of claim 38, wherein the appropriate plant host cell is a rice suspension culture cell. 40. The method of claim 31, wherein the sugar-deficiency or sugar-free state is a state of sucrose, glucose, or fructose deficiency. 41. The method according to item 31 of the patent application, wherein the gene product is a protein of animal, plant or microbial origin. 42. The method according to the scope of patent application No. 31, in which the selection of angiosperm host cells transformed by the vector is facilitated, and the vector further contains -6- This paper size is applicable to Chinese national standards (CNSM4 specification (210 X 297 male i " 3 (please read the note on the back? Matters and then fill out this page) £ 4. Six, _, please patent Fanyuan has an identification gene, reporter gene, antibiotic-resistance gene, enhancer or regulatory sequence as A colony marker. 43. If the method of the 42nd scope of the patent application, the vector further contains an antibiotic-resistance gene as a colony marker. 44. The method of the 43rd scope of the patent application The antibiotic targeted by the antibiotic_resistance gene is kanamycin or hygromycin. 45 'The method according to item 42 of the patent application, wherein the vector further contains a report Gene is used as a breeding marker. 46. The method according to item 45 of the patent application, wherein the reporter gene is a / 3-glucuronidase gene. Order 47. If a patent is applied for Methods of around 31, wherein the desired protein / 5 - glucuronic anhydrase Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) Μ