CN110904110B - Application of OsHAP3C gene expression reduction in rice variety with shortened heading period and prolonged growth period - Google Patents
Application of OsHAP3C gene expression reduction in rice variety with shortened heading period and prolonged growth period Download PDFInfo
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Abstract
The invention discloses application of OsHAP3C gene expression reduction in cultivation of rice varieties with prolonged growth period after heading period, wherein the rice OsHAP3C gene is used as a target gene, RNAi interference is used for reducing the expression of the OsHAP3C gene in rice, so that the rice varieties with delayed flowering and prolonged growth period are obtained, and the rice variety has important application value in rice production practice.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to application of OsHAP3C gene expression reduction in cultivation of rice varieties with shortened heading period and prolonged growth period.
Background
Rice (Oryza sativa L.) is one of the most important cereal crops in the world, and is expanded from the south China to wide temperate and subtropical regions, and the heading date is the most important character of rice adapting to different ecological formations. In rice production, the heading period generally directly determines the growth period of rice varieties. The heading stage of rice is mainly influenced by external environmental factors such as self genetic genes, illumination and the like. At present, researches find that the genes controlling the heading stage of rice mainly comprise two types: quantitative Trait Loci (QTL) and major genes. At present, more than 600 QTLs related to the heading stage of rice are distributed on each chromosome of the rice respectively, namely the maximum chromosome 3, the second chromosomes 1, 7 and 8 and the minimum chromosome 10. So far, more than 20 genes influencing the heading stage of rice have been cloned, and mainly directly or indirectly regulate and control the expression of Hd3a and RTF1 to play a role, so that two main regulation approaches are formed: OsGI-Hd1-Hd3a and OsGI-Ehd1-Hd3a/RFT 1. These heading stage regulatory genes also include a class of HAP (HAP) complexes known as transcription factors.
In plants, the HAP complex is composed of 3 subunits, including HAP2, HAP3, and HAP5, each of which presents a gene family of multiple genes. In the rice genome, there are 10 HAP2 genes, 11 HAP3 genes and 13 HAP5 genes. Through the combination of a conserved specific sequence and a CCAAT frame of a eukaryotic promoter region, the expression of downstream genes is regulated and controlled through the interaction with a regulation factor. The HAP3 gene is involved in regulating many important physiological processes of plants, including embryonic development, chlorophyll biosynthesis, drought stress and other physiological processes, and plays an important role in flowering phase regulation.
With the rapid development of molecular biology, it has become a very simple matter to directionally regulate the expression of a certain target gene and further change the agronomic traits. For example, to reduce the expression of a gene, there are various methods and approaches in which RNA interference (RNAi) is a molecular biological technique for blocking the expression of a gene, which specifically and efficiently degrades mRNA transcribed from the DNA of the target gene, thereby regulating the expression of the gene. RNAi is a survival mechanism for regulating gene transcription of many organisms including plants and adapting to external environment, and RNAi interference technology developed based on the mechanism is not only used for researching gene functions, but also has wide application prospects in disease treatment and creation of new crop germplasm.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of reducing the expression of OsHAP3C gene in breeding rice varieties with shortened heading period and prolonged growth period; the second object of the present invention is to provide a method for reducing the expression of OsHAP3C gene in rice; the invention also aims to provide a method for cultivating the rice variety with the heading period pushed back and the growing period prolonged.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the application of the OsHAP3C gene expression reduction in breeding rice varieties with shortened heading period and prolonged growth period, wherein the nucleic acid sequence of the OsHAP3C gene is shown as SEQ ID No. 15.
In a preferred embodiment of the present invention, the method for reducing the expression of the OsHAP3C gene is any method capable of reducing the expression of the OsHAP3C gene, including gene editing and deleting OsHAP3C, preferably an RNAi interference technology and an shRNA technology for forming siRNA.
Preferably, the nucleotide sequence of the interference fragment interfered by RNAi is shown in SEQ ID NO. 1.
2. A method for cultivating a rice variety with a post heading period and an extended growth period specifically comprises interfering OsHAP3C gene expression in rice, wherein a nucleic acid sequence of the OsHAP3C gene is shown as SEQ ID No. 15.
According to the preferred RNAi interference method, an interference vector is constructed by an interference fragment shown in SEQ ID NO.1, agrobacterium is transformed to obtain engineering bacteria, the obtained engineering bacteria are transformed into callus of mature embryos of rice by an agrobacterium-mediated method, the transformed callus is subjected to co-culture, screening, differentiation, seedling strengthening and transplantation, and a rice variety with a heading stage pushed and a growth stage prolonged is obtained through molecular identification.
Preferably, the interference vector is constructed by the following method: amplifying an interference fragment shown in SEQ ID NO.1 by taking sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers and rice Kasalath cDNA as a template, and then connecting an interference vector pYLRNAi.5 through Sac I and Hind III to obtain an intermediate vector pYLRNAi-3C-1-F containing a forward insertion fragment; then, an interference fragment is amplified by taking the intermediate vector pYLRNAi-3C-1-F as a template and the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers, and the interference fragment is connected to the intermediate vector pYLRNAi-3C-1-F through Mlu I and Pst I to obtain the interference vector pYLRNAi-3C-1.
Preferably, the agrobacterium is agrobacterium LBA 4404.
The invention has the beneficial effects that: the RNAi vector is designed and constructed by utilizing the nucleotide sequence shown in SEQ ID NO.1 and aiming at the rice gene OsHAP3C (SEQ ID NO.15), and the expression of the gene can be effectively reduced by transferring the RNAi vector into rice, so that the effects of delaying the heading and flowering of the rice and prolonging the growth period of the rice are realized, and the RNAi vector has important significance for cultivating rice varieties with prolonged growth period after heading period is pushed.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows a partial electrophoretogram of the pYLRNAI-3C-1 vector construct (A: i-3C-1 first PCR amplification product; B: i-3C-1 second PCR amplification product; C: MluI + PstI digestion of plasmid pYLRNAI-3C-1-F; D: LBA4404-pYLRNAI-3C-1 bacterial detection).
FIG. 2 is a partial electrophoretogram of the pYLRNAI-3C-2 vector construct (A: the first PCR amplification product of i-3C-2; B: the second PCR amplification product of i-3C-2; C: the restriction enzyme digestion result of MluI + PstI on the plasmid pYLRNAI-3C-2-F; D: the bacterial detection result of LBA 4404-pYLRNAI-3C-2).
FIG. 3 is a schematic diagram of pYLRNAI-3C vector.
FIG. 4 shows the hygromycin resistance gene (HptII) PCR detection electrophoretogram of RNAi transgenic rice plant (M: marker of DL 2000; P: positive control, pYLRNAI-3C plasmid as template; N: negative control, non-transgenic rice Kasalath total DNA as template; and DNA of 1-7: 7 different transgenic plants as template).
FIG. 5 shows the PCR detection electrophoresis of RNAi fragments of RNAi transgenic rice plants (M: marker of DL 2000; P: positive control, pYLRNAI-3C plasmid as template; N: negative control, non-transgenic rice Kasalath total DNA as template; and DNA of 1-7: 7 different transgenic plants as template).
FIG. 6 shows PCR detection of OsHAP3C gene expression of pYLRNai-3C-1 transgenic rice plants (WT: non-transgenic rice Kasalath RNA as template; RNA of 1-6: 6 different transgenic plants as template).
FIG. 7 shows PCR detection of OsHAP3C gene expression of pYLRNai-3C-2 transgenic rice plants (WT: non-transgenic rice Kasalath RNA as template; RNA of 1-6: 6 different transgenic plants as template).
FIG. 8 shows pot culture experiments of pYLRNAI-3C-2 transgenic rice (WT: wild type; I3C-2: RNAi transgenic rice).
FIG. 9 shows the growth of the pYLRNAI-3C-1 transgenic rice and the wild type control heading flowering (A: potted plant laboratory results; B: field experimental results, WT: wild type; I3C-1: RNAi transgenic rice).
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiment is only an example of the implementation of the present invention, and not all, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention based on the description of the embodiment. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
Example 1 cloning of OsHAP3C Gene interference fragment
1) Extraction of rice seedling RNA: taking 0.1g of leaf of rice Kasalath seed germinating for about 15 days, adopting TRNzol-A+Total RNA was extracted with a reagent (Tiangen Biochemical technology Co., Ltd.). The reference manufacturer operates with instructions:
putting a fresh rice plant leaf sample into a 1.5ml EP tube, immediately putting the EP tube into liquid nitrogen for freezing, and storing the fresh rice plant leaf sample in a refrigerator at the temperature of minus 80 ℃;
secondly, taking out the frozen sample EP tube, adding liquid nitrogen to grind the sample into fine powder;
③ adding 1ml TRNzol-A+Mixing, standing at room temperature for 5 min;
adding 0.2ml of chloroform, oscillating for 15sec, and standing for 2min at room temperature;
fifthly, centrifuging at 4 ℃, 12000g for 15min, and taking the supernatant into a new 1.5mL EP tube;
sixthly, adding isopropanol with the same volume, gently mixing the liquid in the tube, and standing for 15min at room temperature;
seventhly, centrifuging at 4 ℃, 12000g for 10min, and removing supernatant;
adding 1ml of 75% ethanol, and gently washing and precipitating for 2-3 times;
ninthly, centrifuging at 4 ℃, 7500g for 5min, and removing the supernatant;
drying at 37 deg.C, adding 50-100 μ l RNase free dH2And dissolving the O.
2) Obtaining a first strand of reverse transcription cDNA: the reverse transcription kit PrimeScript from Takara was usedTMRT reagent Kit with gDNA Eraser (cat # RR047A), using the extracted rice RNA as template to synthesize the first strand of cDNA, and storing the synthesized cDNA at-20 ℃ for later use.
3) PCR amplification of interfering fragments: based on the coding region sequence and the non-coding region sequence of the nuclear transcription factor OsHAP3C gene, two sequences of about 200bp are selected as RNAi target sequences, the amplification primers of an interference fragment 1(SEQ ID NO.1) are SEQ ID NO.3 and SEQ ID NO.4, the amplification primers of an interference fragment 2(SEQ ID NO.2) are SEQ ID NO.5 and SEQ ID NO.6, and the primer DNA fragments are biosynthesized by Chengdu engine. The reverse transcription cDNA is taken as a template, SEQ ID NO.3 and SEQ ID NO.4 are taken as primers to carry out PCR amplification on the interference fragment 1, and SEQ ID NO.5 and SEQ ID NO.6 are taken as primers to carry out PCR amplification on the interference fragment 2. The PCR reaction was performed in a total volume of 50. mu.L, prepared according to the manufacturer's instructions. PCR amplification procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for 30sec, 35 cycles; extension was continued for 6min at 72 ℃. The PCR product was separated by electrophoresis on a 1.5% agarose gel, and the amplified fragment was recovered and purified by using a DNA gel recovery kit (Tiangen Biochemical technology Co., Ltd.).
Example 2 construction of RNAi vectors and obtaining of engineered bacteria
1) Obtaining of intermediate vector: carrying out double enzyme digestion on the products of the recovered and purified interference fragment 1 and the interference fragment 2 by using Sac I and Hind III respectively, and recovering to obtain a target fragment after enzyme digestion; at the same time, the interference vector pYLRNai.5 is subjected to double enzyme digestion by Sac I and Hind III, and the vector framework fragment is recovered. Connecting a target fragment and a vector, transforming Escherichia coli DH5 alpha, identifying positive clones, sequencing, and obtaining plasmids with correct sequencing, namely the constructed intermediate vector containing the positive insert, which are respectively named pYLRNAI-3C-1-F and pYLRNAI-3C-2-F.
2) Obtaining of interference vector: carrying out PCR amplification by respectively taking plasmids of the intermediate vectors pYLRNAi-3C-1-F and pYLRNAi-3C-2-F as templates and taking SEQ ID NO.7 and SEQ ID NO.8 as primers to obtain an interference target fragment with Mlu I at the upstream and Pst I enzyme cutting site at the downstream, carrying out double enzyme cutting by using Mlu I and Pst I, and recovering the target fragment; simultaneously, Mlu I and Pst I are used for carrying out double enzyme digestion on the intermediate vectors pYLRNai-3C-1-F and pYLRNAI-3C-2-F respectively, and the vector framework fragment is recovered. Connecting the target fragment with the vector skeleton fragment, transforming Escherichia coli DH5 alpha, identifying positive clone, sequencing, and obtaining the final RNAi interference vector containing forward and reverse insertion fragments, named pYLRNAI-3C-1 and pYLRNAI-3C-2, wherein the electrophoresis results of the construction process are shown in FIG. 1 and FIG. 2, and the vector structure diagram is shown in FIG. 3.
3) Obtaining engineering bacteria: firstly, the agrobacterium rhizogenes competence is prepared, and the operation steps are as follows: taking YEP +40 mg.L for agrobacterium LBA4404 stored at-70 ℃ in the laboratory-1Rif+50mg·L-1The single colony of Str solid culture medium is first activated, and then the single colony is picked up and used as YEP +40 mg.L-1Rif+50mg·L-1Str broth activation to second OD600The strain can be used for preparing agrobacterium tumefaciens competent cells by 0.3-0.5: carrying out ice bath on the bacterial liquid for 30 min; ② taking 1mL of bacterial liquid to put in a 1.5mL of EP tube, centrifuging for 5min at 4 ℃ and 5000rpm, and removing supernatant; ③ using sterilized 0.1M CaCl2(precooling) 400 mu L of suspended thallus (lightly beating evenly by using a pipette gun); fourthly, placing the mixture on ice for 30min, centrifuging the mixture for 5min at the temperature of 4 ℃ and 5000rpm, and removing supernatant; fifthly, pre-cooling 0.1M CaCl with 50-100 mu L2Suspending thallus, storing at 4 deg.C for 10 hr or adding 20-30% sterilized glycerol, mixing, quick freezing with liquid nitrogen, and storing at-70 deg.C.
The constructed recombinant plasmids pYLCNai-3C-1 and pYLCNai-3C-2 are introduced into the prepared agrobacterium LBA4404 competent cells by a freeze-thaw method: taking 10 mu L (3-5 mu g) of recombinant plasmid, adding the recombinant plasmid into 100 mu L of LBA4404 competent cells in a tube, and gently mixing the recombinant plasmid and the competent cells; ② immediately placing on ice for 30min, and quickly putting into liquid nitrogen for 2 min; thirdly, rapidly putting the mixture into water bath at 37 ℃ until the mixture is completely melted; fourthly, adding 800 mu L of YEP liquid culture medium without antibiotics, and gently mixing the materials; shaking culture and activating (28 deg.C, 200rpm, 3-5 h); fifthly, centrifuging at room temperature (4000rpm for 5min), removing supernatant, and mixing the residual 100 mu L of bacterial liquid with suspended thallus; sixthly, evenly coating 100 mu L of heavy suspension activated bacteria liquid to YEP plus 40 mg.L-1Rif+50mg·L-1Str+100mg·L-1Kan solid screening cultureOn a substrate plate; seventhly, carrying out inverted culture in a 28 ℃ culture box for 2-3 days until the grown bacterial colony is moderate in size, and selecting resistant single bacterial colony containing YEP +40 mg.L-1Rif+50mg·L-1Str+100mg·L-1Culturing in Kan liquid culture medium. After the culture bacterial liquid reaches a certain concentration, PCR detection is carried out (the PCR system and conditions for detecting the target gene are the same as the above). The constructed engineering bacteria are named as LBA4404-pYLRNAI-3C-1 and LBA 4404-pYLRNAI-3C-2.
Example 3 genetic transformation of Rice by engineering bacteria
An agrobacterium tumefaciens mediated method is adopted, an interference vector is introduced into rice callus, a transgenic plant is obtained by screening hygromycin B, a basic culture medium N6 and MS cultures are purchased from Phytotechnology Laboratories, and the formula and the preparation method of the culture medium are as follows:
first, an Infection Medium (IM): n6 salt +0.7 g.L-1L-proline +68.4 g.L-1Sucrose +36 g. L-1Glucose +1.5 mg. L -12,4-D+2mg·L-1Glycine +1 mg. L-1VB1+0.5mg·L-1VB6+0.5mg·L-1Nicotinic acid, pH 5.2, filter sterilized with 0.22 μ M filter, stored at 4 ℃ and added with 100 μ M Acetosyringone (AS) immediately before use.
② Callus Induction Medium (CIM): n6 salt +2.8 g.L-1L-proline +30 g.L-1Sucrose +300 mg. L-1Hydrolyzed casein +2 mg. L -12,4-D+4g·L-1Adjusting pH of the plant gel to 5.8, sterilizing at 121 deg.C for 20min, adding 2 mg/L-1Glycine +1 mg. L-1VB1+0.5mg·L-1VB6+0.5mg·L-1Nicotinic acid.
③ Co-Culture Medium (CM) (+ 10 g.L) N6 salt-1Glucose +30 g. L-1Sucrose +300 mg. L-1Hydrolyzed casein +2 mg. L -12,4-D+4g·L-1Adjusting pH of the plant gel to 5.8, sterilizing at 121 deg.C for 20min, adding 2 mg/L-1Glycine +1 mg. L-1VB1+0.5mg·L-1VB6+0.5mg·L-1Niacin +100 μ M AS.
SieveSelection Medium (SM): CIM +250 mg. L-1Carbenicillin (Cb) +50 mg. L-1Hygromycin B (Hyg).
Fifth differentiation Medium I (Regeneration Medium I, RMI): MS salt +30 g. L-1Sorbitol +30 g.L-1Sucrose +2 g.L-1Hydrolyzed casein +2 mg. L-1Hormone (KT) +0.02 mg. L-1alpha-Naphthylacetic acid (NAA) +4 g.L-1Adjusting pH of the plant gel to 5.8, sterilizing at 121 deg.C for 20min, adding 2 mg/L-1Glycine +1 mg. L-1VB1+0.5mg·L-1VB6+0.5mg·L-1Nicotinic acid +100 mg. L-1Cefprocillin (Cef) +10 mg.L-1Vancomycin (Van) +50 mg. L-1Hyg。
Sixthly, Rooting Medium (RM): MS salt +2 mg. L-1Glycine +1 mg. L-1VB1+0.5mg·L-1VB6+0.5mg·L-1Nicotinic acid +100 mg. L-1Inositol +30 g.L-1Sucrose +5 g.L-1Agar powder, pH 5.8.
The genetic transformation procedure was as follows:
firstly, induction culture of rice mature embryo callus: soaking the hulled mature rice seeds in ethanol with the volume fraction of 70% for 30 seconds, then soaking the seeds in 0.1% mercuric chloride for 8-10min, performing surface sterilization, washing the seeds with sterile water for 7-8 times, placing the seeds on sterile filter paper to absorb water, and then placing the seeds on a mature embryo callus induction culture medium CIM, wherein the illumination period is as follows: light illumination at 33 deg.C for 14h, and dark culture at 30 deg.C for 10 h. After about 10-15 days, the callus grown from the scutellum of the mature embryo is peeled off, transferred to a mature embryo subculture medium CIM, and subcultured under the same conditions. Subcultured every two weeks thereafter. Three days before the infection, the callus was cut into 2-4 mm size and transferred to a new induction medium for preculture.
② culturing and preparing agrobacterium: agrobacterium LBA4404-pYLRNai-3C-1 and LBA4404-pYLRNai-3C-2 were separately added at LB +50 mg.L-1Str+100mg·L-1Streaking was performed on Kan plates, and dark culture was performed at 19 ℃ for three days. A small amount of the bacteria are picked up by an inoculating loop and suspended in 15 ml of a staining culture medium IM +100 mu M AS, and the concentration OD of the bacteria is adjusted5500.06-0.08, i.e.Agrobacterium suspensions for transformation of rice.
③ impregnating and co-culturing agrobacterium: the pre-cultured rice callus is inoculated into 15 ml of dip-dyeing culture medium and 100 mu M acetosyringone for pre-washing once, liquid is poured out, 15 ml of the prepared agrobacterium suspension is poured in, and the mixture is gently shaken for 2 minutes. The bacterial solution was decanted, the callus was placed on sterile filter paper and excess bacterial solution was aspirated, and then transferred to co-culture medium and cultured in the dark at 19 ℃ for three days.
And fourthly, screening the bacterium washing and resistant callus: washing the callus after three days of co-culture with sterile water for 7-8 times, and then staining the culture medium with 250 mg. L-1Carbenicillin +10 mg. L-1Washing vancomycin once, placing the callus on sterile filter paper to remove excess liquid, transferring to screening medium SM (illumination at 33 deg.C for 14h, dark culture at 30 deg.C for 10h), subculturing once every 14 days, and screening for 2 times.
Fifthly, differentiation of the resistant callus: from the resistant callus which grows out after two rounds of screening, the selected cream-yellow compact resistant callus is transferred to a medium RMI containing differentiation, green spots appear in about 14-20 days, and the cream-yellow compact resistant callus is further differentiated into plantlets in about 30 days.
Sixthly, rooting, strengthening and transplanting of the transformed seedlings: when the bud differentiated from the resistant callus grows to about 2-4 cm, the plantlet is transferred to a rooting culture medium and cultured for about two weeks and then transferred to a strong seedling culture medium for 2-3 weeks. Selecting plantlets with height of about 10 cm and developed root system, washing off culture medium with warm water, and transplanting in the greenhouse. The water surface is not submerged, if the weather is fine, the seedlings need to be shaded until the seedlings survive (based on water spitting).
Example 4 screening and identification of transgenic Rice
1) PCR detection of transgenic plants: for the transformed plant obtained in example 3, genomic DNA was extracted by CTAB method, and conventional PCR detection was performed on hygromycin resistance gene Hpt II (amplification primers SEQ ID NO.9 and SEQ ID NO.10) and RNAi fragment (amplification primers SEQ ID NO.7 and SEQ ID NO.8), respectively, and PCR amplification was performed under the same conditions as positive and negative controls using plasmid pYLRNai-3C-1 and non-transgenic rice Kasalath total DNA as templates, respectively. The transgenic positive plant is a plant in which both Hpt II and RNAi fragments are amplified to be positive, but the non-transgenic rice Kasalath cannot generate a strip after amplification. The results of PCR electrophoretograms of the HptII gene and the RNAi fragment are shown in FIGS. 4 and 5. The results show that HptII genes and RNAi fragments can be detected in No. 1-7 plants, and the detected 7 transformed plants are transgenic positive plants.
2) RT-PCR detection of gene OsHAP3C expression: transgenic plants identified as positive by PCR in step 1) of example 4 were used for total RNA extraction, while non-transgenic wild-type Kasalath was used as control material. The total RNA is reverse transcribed to obtain cDNA, the cDNA is taken as a template, OsHAP3C gene specific primers SEQ ID NO.11 and SEQ ID NO.12 are used for amplification, the total volume of PCR reaction is 25 mu L, and the preparation is carried out according to the instructions of manufacturers. The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 30sec, 36 cycles; extension at 72 ℃ for 5 min. The PCR product was visualized by 1.5% agarose gel electrophoresis. Meanwhile, rice Ubiquitin gene (UBQ) is used as an internal reference for amplification, primers are SEQ ID NO.13 and SEQ ID NO.14, and a PCR amplification program is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 30sec, 28 cycles; extension at 72 ℃ for 5 min. The detection result shows that in the transgenic plants transferred with pYLRNAI-3C-1, some DNA fragments which can not detect the specific amplification of the OsHAP3C gene are weak bands compared with the wild type (partial results are shown in figure 6), and the detection result proves that after the interference fragment 1 is transferred, the expression of the OsHAP3C gene is interfered, and the expression of the OsHAP3C gene in some plants is reduced. In the transgenic plants of the pYLRNAI-3C-2, the expression of the OsHAP3C gene in all the detected plants is basically consistent with that of the wild type of the control, and the difference is not obvious (partial results are shown in figure 7), which indicates that the transferred interference fragment 2 does not have the interference effect on the OsHAP3C gene of the target gene.
Example 5 phenotypic characterization of transgenic plants
Observation of the 'Kasalath' phenotype of OsHAP3C RNAi transgenic rice and wild-type rice:
(1) the transgenic plant transferred with pYLRNAI-3C-2 has no obvious difference with the wild type in the aspects of phenotype and agronomic traits (as shown in figure 8), and the main reason is that the RNAi interference vector constructed by the nucleotide sequence shown in SEQ ID NO.2 does not interfere with the target gene OsHAP 3C.
(2) Compared with wild rice 'Kasalath', the heading period of transgenic rice with reduced OsHAP3C RNAi expression is obviously delayed by about 6-10 days and the whole growth period is prolonged by about 6-10 days (as shown in figure 9) in both pot experiment and field experiment of transgenic plants of the interference vector pYLRNAI-3C-1 constructed by the nucleotide sequence shown in SEQ ID NO. 1. The field agronomic trait investigation was carried out for two consecutive years (2018 and 2019) on the RNAi transgenic line with reduced expression of OsHAP3C, and the results are shown in tables 1 and 2.
TABLE 1, 2018 agronomic character questionnaire for pYLRNAI-3C-1 transgenic and non-transgenic plants
TABLE 2 survey table of agronomic characters of plants and non-transgenic plants of pYLRNAI-3C-1 transgenic plants in 2019
From the investigation result, the plant height of the RNAi transgenic rice is slightly shorter than that of the control wild type non-transgenic rice, the number of grains per spike is smaller than that of the control, but the effective spike number and the spike length are generally increased, and the hundred grain weight is slightly reduced compared with that of the control. After the OsHAP3C gene is interfered, the growth period of the rice can be influenced, and the effective ear number and the ear length are slightly influenced.
The results show that the scheme of the invention can achieve the effects of delaying the heading stage of the rice and prolonging the growth period, and has important application value in rice production practice.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> institute of biotechnology and nuclear technology of academy of agricultural sciences of Sichuan province
<120>ReduceOsHAP3CApplication of gene expression in cultivation of rice variety with shortened heading period and prolonged growth period
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 212
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcatagcatc gtctccagcg gagtcaaaca caaagacaag tcgagatccg gcggccggtg 60
gcgtcctcct ccctctccct cctccccaac caacggcgct gatcccctcc gccatctccg 120
tccatctccg cctaaaaaaa ctaagcgatg tcggaggggt tcgacgggac ggagaacggc 180
ggcggcggcg gcggcggagg cggagtaggg aa 212
<210> 2
<211> 184
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tggggacgac ctgatctggt caatgggcac gctcggattc gaggactatg tcgagcctct 60
caagctctac ctcaggctct accgggagac ggagggtgac acaaagggtt caagagcttc 120
tgaactgcca gtaaagaaag atgttgtact taatggagat cctggatcat cgtttgaagg 180
catg 184
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cagagctcgc atagcatcgt ctccag 26
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cacaagcttt tccctactcc gcctccg 27
<210> 5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cagagctctg gggacgacct gatctg 26
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
caccctgacg cgtggtgtta cttctgaaga gg 32
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
actagaactg cagcctcaga tctaccatgg tcg 33
<210> 9
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgatttgtgt acgcccgaca gtc 23
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<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgatgtagga gggcgtggat atg 23
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<213> Artificial Sequence (Artificial Sequence)
<400> 11
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<213> Artificial Sequence (Artificial Sequence)
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ctacatgcct tcaaacgatg atc 23
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<213> Artificial Sequence (Artificial Sequence)
<400> 13
accctggctg actacaacat c 21
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<213> Artificial Sequence (Artificial Sequence)
<400> 14
agttgacagc cctagggtg 19
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<213> Rice (Oryza sativa L.)
<400> 15
atgtcggagg ggttcgacgg gacggagaac ggcggcggcg gcggcggcgg aggcggagta 60
gggaaggagc aggaccggtt cctgccgatc gccaacatcg gccgcatcat gcgccgggcc 120
gtgccggaga acggcaagat cgccaaggac tccaaggagt ccgtccagga gtgcgtctcc 180
gagttcatca gcttcatcac cagcgaagca agcgacaagt gcctcaagga gaagcgcaag 240
accatcaatg gggacgacct gatctggtca atgggcacgc tcggattcga ggactatgtc 300
gagcctctca agctctacct caggctctac cgggagacgg agggtgacac aaagggttca 360
agagcttctg aactgccagt aaagaaagat gttgtactta atggagatcc tggatcatcg 420
tttgaaggca tgtag 435
Claims (4)
1. ReduceOsHAP3CApplication of gene expression in cultivating rice variety with heading stage after heading stage and growth stage prolonging, wherein the gene expression is used for promoting growth of rice varietyOsHAP3CThe nucleic acid sequence of the gene is shown as SEQ ID NO. 15; said reductionOsHAP3CThe gene expression method adopts RNAi interference capable of forming siRNA; the nucleotide sequence of the interference fragment interfered by RNAi is shown as SEQ ID NO. 1.
2. A method for cultivating a rice variety with a shortened heading period and an extended growth period is characterized by comprising the following steps: in particular by interference in riceOsHAP3CGene expression of saidOsHAP3CThe nucleic acid sequence of the gene is shown as SEQ ID NO. 15; the RNAi interference method is characterized in that an interference vector is constructed by an interference fragment shown in SEQ ID NO.1, agrobacterium is transformed to obtain engineering bacteria, the obtained engineering bacteria are transformed into callus of mature embryos of rice by adopting an agrobacterium-mediated method, the transformed callus is subjected to co-culture, screening, differentiation, seedling strengthening and transplantation, and a rice variety with a shortened heading period and an extended growth period is obtained through molecular identification.
3. The method of claim 2, wherein: the interference vector is constructed by adopting the following method: the sequence shown in SEQ ID NO.3 and SEQ ID NO.4 is used as a primer, the rice Kasalath cDNA is used as a template to amplify the interference fragment shown in SEQ ID NO.1, and the amplification is carried out bySac 
AndHind 
connecting an interference vector pYLRNai.5 to obtain an intermediate vector pYLRNai-3C-1-F containing the forward insert; then using an intermediate vector pYLRNai-3C-1-F as a template and the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers to amplify the interference fragment, and carrying out amplification byMlu 
AndPst 
the intermediate vector pYLRNai-3C-1-F is connected to obtain the interference vector pYLRNai-3C-1.
4. The method of claim 3, wherein: the agrobacterium is agrobacterium LBA 4404.
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