CN103421805A - Cloning and application of major gene qpc1 controlling protein content of rice endosperm - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering and discloses a major gene qpc1 controlling the protein content of rice endosperm and application of an allele of the major gene qpc1. The sequence of the major gene qpc1 is shown in SEQ ID NO:1, the sequence of the allele of the major gene qpc1 is shown in SEQ ID NO:2. A prediction shows that the protein of the major gene qpc1 is an amino acid transmembrane transport protein with a PF01490 conserved domain. Through comparison of sequencing, 23 common base differences exist between two varieties in the range of 9.9 kb, wherein 15 mutations are located in a promoter region, 7 mutations are located in a first intron region, 1 mutation is located in a coding region, but the coding of amino acid sequences is not changed. Through the transgenosis, paddy rice plants with the transferred major gene qpc1 are obtained. The protein contents of the seeds of both overexpression, complementary and transformational transgenic plants are improved remarkably when compared with those of reference seeds, but the protein contents of the reference seeds of transgenic plants of which the RNAi is suppressed in expression are lowered remarkably when compared with those of the reference seeds.

Description

Control the major gene qpc1 clone and application of paddy endosperm protein content

Technical field

The present invention relates to Plant Biotechnology and gene engineering technology field.Be specifically related to gene clone and an application that is positioned at the main effect QTL (qpc1) of the long-armed upper control paddy endosperm protein content of paddy rice the first chromosome.

Background technology

The paddy endosperm protein content is an important economical character: along with the opening of standard of living raising and Chinese rice market, people are also more and more higher to the requirement of the quality of rice.The physical and chemical index of edible high quality rice generally comprise processing quality, exterior quality, cooking and eating quality and 4 main aspects of nutritional quality (Zhang etc., 2007, Proc.Natl.Acad.Sci.U.S.A.104:16402-16409).The nutritional quality of rice depends mainly on the height of protein, Methionin and part vitamin contents in polished rice.And protein is one of most important nutritional quality of paddy rice, its content in rice occupy second, is only second to starch, is the main source that people obtain protein.In cereal crop, it is relatively good that the protein of rice forms, because gluten accounts for more than 80% of rice total protein content, and Methionin mainly is present in gluten; And the amino acid of gluten forms relatively rationally, be mainly that methionine content is higher, this is the unexistent advantage of other plant, be acknowledged as the high-quality food protein (Yan Qichuan, 2001, seed science. Beijing: Chinese agriculture press publishes, 58-59).With other albumen by contrast, rice protein is safest a kind of albumen up to now, rice be unique a kind of can not need just can be directly edible through hypersensitive test food crop (Wang Wen is high, 2001, grain and grease .5:32-33).Improving constantly of understanding rice protein is worth along with people, take rice protein as raw material, is developed to food that added value is very high and other some additive products.Such as the hi-protein nutritious cereal of selling on market, flavor peptides etc., also having the flavour of food products modifying agent of selling on market is exactly that rice protein hydrolysis post-treatment forms, generally obtain people's approval (Wang Zhangcun etc., 2004, Chinese grain and oil journal .2:1-15).

The hereditary basis more complicated of paddy endosperm protein content is typical quantitative character.Utilize the QTL (Quantitative Trait Loci) that molecular marking technique can paired domination number amount proterties position and decompose, complicated quantitative character is decomposed into to simple Mendelian factor and is studied.Utilize this method, had been found that in recent years the QTLs of many control Protein Content of Rices, and the location of these QTLs is scarcely out of swaddling-clothes, and most research mainly concentrates on Brown Rice protein content (.2001 such as Tan, Theor.Appl.Genet.103:1037-1045; Aluko etc., 2004, Theor.Appl.Genet.109:630-639; Yu Yonghong etc., 2006, Acta Agronomica Sinica .32 (11): 1712-1716; Li Chen etc., 2006, plant genetic resources journal .7 (2): 170-174), about the research of polished rice protein content still less (Zhong Ming etc., 2007, Molecular Plant Breeding .5 (5): 631-638; Weng Jianfeng etc., 2006, Acta Agronomica Sinica .32 (1): 14-19).Therefore qpc1 has huge application potential and prospect for the improvement of rice quality proterties, and the qpc1 gene is cloned new important genetic resources can be provided for the quality breeding of paddy rice.

In heredity, QTL and the same can the separation by genetic recombination of gene of controlling qualitative character, difference is varying in size of hereditary effect, so QTL can carry out Fine Mapping equally.When utilizing primary group to carry out the QTL location, the fiducial interval of QTL is (Darvasi etc. more than 10cM usually, 1992, Theor.Appl.Genet.85:353-359), be difficult to determine that the main effect QTL detected is one or a plurality of minor effect QTL is (Yano and Sasaki on earth, 1997, Plant Mol.Bio.35:145-153).Therefore on the basis of elementary location, QTL is carried out to Fine Mapping (<1cM) and just seem and be necessary very much.And be exactly to build the chromosome segment that contains target QTL to substitute (the Chromosome segment substitution line of system for the best approach of QTL Fine Mapping, CSSL) or near isogenic line (Nearly isogenic line, the secondary target group (Secondary population) such as NIL), make in colony to only have single QTL site to be separated, reduced to the utmost the interference that objective trait is produced caused because genetic background is different with environment between individuality, made this site show as simple Mendelian factor heredity.This method has been brought into play important effect (Frary etc., 2000, Science. 289:85-88 in the Fine Mapping of many QTL and gene clone research; Li etc., 2011, Nature genetics, 43:1266-1269).These methods are for the strategy that adopts map based cloning separates, the gene of clone qpc1 provides foundation.

The present invention utilizes one of the method separating clone of map based cloning to control the major gene qpc1 of paddy endosperm protein content, for the quality breeding of paddy rice provides new genetic resources.

Summary of the invention

The objective of the invention is complete coding region segment DNA fragment of controlling the major gene of paddy endosperm protein content and quality trait of separating clone from paddy rice, utilize the ability of this improvement of genes rice quality.This unnamed gene that the applicant will clone is qpc1.The present invention relates to separate and apply the gene qpc1 of a Brown Rice and polished rice, and the mechanism of action of this fragment is set forth.Wherein, described fragment is as shown in sequence table SEQ ID NO:1 or SEQ ID NO:2, perhaps basically be equivalent to the height homologous DNA sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or its function is equivalent to the subfragment of sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

Select to contain the basically identical material of qpc1 gene and genetic background and Zhenshan 97B the RILs that the present invention accounts for from Zhenshan 97B/Nan Yang (Recombinant inbred lines) colony, with the Zhenshan 97B third backcross generation, selfing, the near isogenic line (Fig. 2) of structure qpc1.Utilize the near isogenic line population analysis, find that the protein content of this gene pairs rice has very large effect (table 1), progeny testing shows, this gene presents Mendelian gene and separates than (Fig. 3).Utilize the hereditary large group of qpc1 near isogenic line and the method for map based cloning, the qpc1 Fine Mapping is arrived in the chromosome segment of 19.8kb (Fig. 4), there are the sequence information of a complete corresponding full-length cDNA, i.e. AK121636 (Rice GenomeAnnotation Proiect Rice Genome Browser-Release 7) in this section.The gene structure of qpc1 and the protein of coding are predicted and analyzed, find that this gene comprises 4 exons, 466 amino acid of encoding altogether, predict that by bioinformatics technique this protein is transmembrane amino acid transporter albumen, has the PF01490 conserved domain.Real-Time PCR expression analysis is found, this gene has expression the time of infertility in each tissue, and the expression amount high (Fig. 5) of the kind (as Zhenshan 97B) that grain protein content is high in the clever shell endosperm kind (as Nan Yang account for) lower than protein content.Prediction is expressed and is measured keying action, so utilize round pcr to obtain the clone of Zhenshan 97B, after subclone, carries out the transgenosis overexpression, and RNAi suppresses to express and the checking of complementary transformation experiment, in transgenosis _T0 generation and T ₁Just in time contrary with protein content in positive individual plant is expressed in the RNAi inhibition for the situation in positive overexpression plant, and the complementary T that transforms ₀In generation,, positive individual plant all showed endosperm protein content existence rising (Fig. 6) in various degree, and at T ₁For in plant they phenotype and genotype be also be divided into from (Fig. 7).In addition, utilize the kind that the kind that grain protein content is high (as Zhenshan 97B) and protein content are low (as Nan Yang accounts for) to compare order-checking, discovery exists 23 common SNP and Indel difference in the promotor of 1.8kb and 4.1kb coding region scope between two kinds, wherein 15 sudden changes are positioned at promoter region, 7 sudden changes occur in the 1st intron zone, 1 sudden change is positioned at the 4th exon of coding region, but not causing amino acid to change, is therefore same sense mutation (Fig. 8).The variation that these results suggest that promoter region is the reason that causes expression amount to change, and then causes the variation of protein content in paddy endosperm, illustrates that qpc1 is the gene of protein content in a positive adjusting and controlling rice endosperm simultaneously.

The invention has the advantages that:

(1) the present invention has cloned a gene that protein content in rice is had to very large positive regulating effect first in paddy endosperm, this quality breeding that is paddy rice provides new genetic resources, also for clone's genes involved in other crop, provides technological borrowing;

(2) gene of cloning in the present invention also can be produced evidence for the molecular evolution research of the dicotyledonous crops such as the cereal crop such as paddy rice and rape.

More detailed technical scheme is as described in " embodiment ".

The accompanying drawing explanation

Sequence table SEQ ID NO:1 is the nucleotide sequence (wherein comprising its corresponding aminoacid sequence) of qpc1 major gene of the source Zhenshan 97B of separating clone of the present invention, and order sequence total length is 5935bp.

Sequence table SEQ ID NO:2 is the allelic nucleotide sequence associated with SEQ ID NO:1 major gene (wherein comprising its corresponding aminoacid sequence) that derives from the qpc1 that Nan Yang accounts for of separating clone of the present invention, and order sequence total length is 5941bp.

Fig. 1: be techniqueflow chart of the present invention.

Fig. 2: be that near isogenic line of the present invention builds schema.While backcrossing, using Zhenshan 97B as female parent.

Fig. 3: BC ₃F ₂The frequency distribution of grain protein content in random population.

Fig. 4: be the performance of protein content of near isogenic line of the present invention and the map based cloning of qpc1 gene.The restructuring number of times occurred between each mark of the digitized representation between mark and qpc1 site.The restructuring number of times occurred between the restructuring individual plant that the digitized representation in bracket screens in first large group and qpc1 site.

Fig. 5: the qpc1 gene is the expression level schematic diagram in the rice at whole growth periods different tissues.Using material is NIL (ZS97, i.e. precious Shan 97) and NIL (NYZ, Nan Yang accounts for) qpc1 gene pure material.In figure, the mark of X-coordinate is as follows: RO is root, and SY is stem, and YL is sword-like leave, and FL is spire, and YP is young fringe, and H4H is the young fringe before blooming four days, and HOH is the clever shell of blooming the same day, and H2A is the seed of blooming latter 2 days, and 5E is the endosperm after blooming 5 days.

Fig. 6: T of the present invention ₀Generation and T ₁Performance and phenotype statistical study for the protein content of transgenosis individual plant.Fig. 6 A is T ₀For the statistics of the protein content of transgenosis individual plant, the ZH11 in Fig. 6 A brace spends 11 in paddy rice; NYZ is that Nan Yang accounts for.Fig. 6 B is T ₁For the statistics of the protein content of transgenosis and near isogenic line individual plant thereof, parenthetic ZH11 spends 11 in paddy rice; NYZ is that Nan Yang accounts for; ZS97 is precious Shan 97; NILs is near isogenic line.

Fig. 7: T of the present invention ₁For the genotype of individual plant in the transgenosis family and being divided into from detected result of phenotype.Wherein black is the transgenic positive individual plant, and white represents transgenosis negative control individual plant.

Fig. 8: structural representation and its natural variation of the present invention clone's qpc1 gene are analyzed.The square frame of black represents exon, fine rule represents intron, " ATG " and " TGA " is respectively translation initiation password and termination codon, fine rule before ATG represents the promotor of 1.8kb, 23 SNP between two parents (Zhenshan 97B and Nan Yang account for) and InDel variation are positioned at the promotor of qpc1 gene and coding region, and (SNP replaces all and represents by corresponding base, the InDel disappearance is all with corresponding horizontal line representative), coding region has 1 base to undergo mutation, but coding same amino acid (arginine).

Fig. 9: the collection of illustrative plates of double base overexpression vector pCAMBIA1301S (carrier is so kind as to give by Australian CAMBIA laboratory).

Figure 10: the collection of illustrative plates of binary expression vector pCAMBIA1301 (transformation on the basis of the pCAMBIA1301S carrier that this carrier is so kind as to give by Australian CAMBIA laboratory).

Figure 11: the collection of illustrative plates that suppresses expression vector ds-pCAMBIA1301 (ds1301).(transformation on the basis of the double base overexpression vector pCAMBIA1301S that this carrier is so kind as to give by Australian CAMBIA laboratory).

Embodiment

Technological line according to Fig. 1, account for RIL (RILs) F8 for selecting to contain the basically identical family of qpc1 gene and genetic background and Zhenshan 97B colony from Zhenshan 97B and Nan Yang, with Zhenshan 97B, hybridize, take Zhenshan 97B as recurrent parent, 3 generations of continuous backcross, selfing, built the near isogenic line of paddy rice qpc1 gene.Utilize a BC who contains 320 strains ₃F ₂Random microcommunity has been carried out location and hereditary effect analysis to qpc1; Utilize the SSR mark of target interval and the insertion/deletion mark (being shown in Table 1) of design to screen the restructuring individual plant to the large genetic group that comes from 10008 individual plants, qpc1 navigates to PB11 and PB12 interval the most at last, and both physical distances are 19.8kb (seeing Fig. 4); Then in conjunction with the gene order information only existed in this section, the gene structure of qpc1 and the protein of coding are predicted and analyzed, find that this gene comprises 4 exons, 466 amino acid of encoding altogether, predict that by bioinformatics technique this protein has the function of amino acid transporter.Real-Time PCR expression analysis is found, this gene time of infertility is at each tissue expression, and in the watery stage and the expression amount of the kind (Zhenshan 97B) that in ripe seed, protein content the is high kind (Nan Yang account for) lower than protein content high.Prediction is expressed and is measured vital role, so utilize the positive colony of round pcr acquisition Zhenshan 97B, after subclone, carries out transgenosis overexpression, RNAi inhibition expression and complementary the conversion and verifies, at transgenosis T ₀Generation and T ₁Just in time contrary with positive individual plant protein content in the RNAi inhibition is expressed for the protein content in positive overexpression individual plant, and the complementary T that transforms ₀In generation,, positive individual plant all showed endosperm protein content existence rising (seeing Fig. 6) in various degree, and at T ₁For in plant they phenotype and genotype be also be divided into from (Fig. 7).In addition, two parents of paddy rice (Zhenshan 97B and Nan Yang account for) are compared to order-checking, discovery exists 23 common SNP and InDel difference in the promotor of 1.8kb and 4.1kb coding region scope between two kinds, wherein 15 sudden changes are positioned at promoter region, 7 sudden changes are positioned at the 1st intron of coding region, and the sudden change of 1 base does not change the amino acid (Fig. 8) of its coding.Because the coding region at qpc1 does not have amino acid whose variation, and then illustrated that the variation of promoter region is the reason that causes expression amount to change, and then caused the variation of endosperm protein content.

Following examples further define the present invention, and have described separating clone qpc1 gene, genetic transformation, the relatively method of sequence difference and this gene spatial and temporal expression spectrum between sequence verification qpc1 allelotrope.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and, in the situation that do not depart from the present invention's essence and scope, can make various changes and modification to the present invention, so that its applicable various uses and condition.

Embodiment 1: build the qpc1 near isogenic line

1, backcross and select

As shown in Figure 2, account for from Zhenshan 97B/Nan Yang that (Zhenshan 97B, be the long-grained nonglutinous rice maintenance line, derives from Jiangxi Province academy of sciences, by Yan Longan researcher, cultivated and to be formed; The researcher of Raleigh army of Nan Yang Zhan You Shanghai City Agricultural biological Gene Center provides) RILs colony in select to contain the basically identical family (RIL-105 of qpc1 gene and genetic background and Zhenshan 97B, compare the genetic background with 68% with Zhenshan 97B), with Zhenshan 97B hybridization, obtain BC ₁F ₁.Then plant lower BC ₁F ₁And again with Zhenshan 97B, backcross once when it is bloomed, obtain BC ₂F ₁.BC under kind ₂F ₁And again with Zhenshan 97B, backcross once when it is bloomed, obtain BC ₃F _i, by BC ₃F ₁Under kind, cross-fertile just obtains BC ₃F ₂.Take Zhenshan 97B as recurrent parent, 3 generations of continuous backcross, and at BC ₁F ₁And BC ₂F ₁In generation, only carried out the forward selection to qpc1, and the select target section is that Zhenshan 97B/Nan Yang accounts for backcross (with molecule marker RM472 and RM104 (in Table 1) target section carried out to forward selection) of the individual plant of heterozygous genes type for next round.The target section with reference to the QTL positioning result determine two SSR (Simple Sequence Repeat) mark RM472 and RM104 (referring to: ( Http:// www.gramene.org/) in the interval defined.At BC ₃F ₁Generation, except carrying out the forward selection, also the genetic background beyond the target section is scanned, therefrom select the offspring (BC of the individual plant that genetic background and Zhenshan 97B are the most close ₃F ₂And BC ₃F ₃) for follow-up experiment.Wherein at BC ₃F ₁Middlely with molecule marker RM472 and RM104, the target section is accredited as to the individual plant of heterozygosis, plants and solidly can obtain near isogenic line Zhenshan 97B (NIL (ZS97)) and the near isogenic line Nan Yang accounts for (NIL (NYZ)) down.

The present invention obtains near isogenic line material paddy rice (Oryza sativa) NIL (ZS97) and delivers China on December 11st, 2012. Wuhan. and the center preservation of Wuhan University's Chinese Typical Representative culture collection, its deposit number is CCTCC NO:P201212.

2.SSR method

The PCR standard program is referring to referring to J. Pehanorm Brooker etc., and 2002, molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), the method that Science Press is introduced.PCR adopts the reaction system of 20 μ l, comprise: the 20-50ng DNA profiling, 10mM Tris-HCl, 50mM KCl, 0.1%Triton X-100,1.8mM MgCl2,0.1mM dNTP, 0.2 μ M primer (primer of RM472 as above and RM104, referring to http://www.gramene.org/) and 1U Taq DNA polymerase.The condition of pcr amplification is: 94 ℃ of denaturation 4min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 34 circulations; 72 ℃ are extended 10min.The PCR product after separating on 6% polyacrylamide gel, carry out silver dye (Bassam etc., 1991, Anal.Biochem.196:80-83).

The Fine Mapping of embodiment 2:qpc1 and map based cloning

1, the measurement of grain protein content phenotype

The grain seasoning is placed under room temperature the relative consistent of water content between dry and each strain of at least placing more than 3 months to guarantee grain.Utilize paddy to go out shelling on rough machine and obtain brown rice, brown rice is ground on rice polisher to polished rice.At the protein content of the upper rapid detection brown rice of near infrared quick analytic instrument (Foss XDS RapidContent Analyzer) and polished rice, method referring to document (Perbandt etc., 2010, BioEnerg.Res.3:194-203).

From BC ₃F ₁The BC that individual plant is derivative ₃F ₂In colony random choose 320 individual plants form a random microcommunity.Investigate Zhenshan 97B, Nan Yang accounts for and random population in the protein content of each individual plant, and determine each pnca gene type with two ends molecule marker RM472 and RM104 (in Table 1), found that this proterties all has significant difference between two parents.In random population, its phenotypic number is divided into groups, make the number of times distribution plan, present approximate bimodal distribution, see Fig. 3.Test in hypothetical 3: 1, v=k-1=1, X are made in height separation to protein content ²On the level of=4.3991, p=0.05, reach remarkable.Show at this BC ₃F ₂In colony, grain protein content is controlled by a major gene, and the low allelotrope allelotrope high to protein content of protein content shows as incomplete recessive function.

2. the development of molecule marker

In the present invention, SSR label information used all comes from Gramene site databases (http://www.gramene.org/).In addition, also according to the genome sequence (http://rise.genomics.org.cn/) of the genome sequence (http://rgp.dna.affrc.go.jp) of the online japonica rice variety Nipponbare announced and rice variety 93-11,12 pairs of Indel (Insert/Deletion) marks that there is polymorphism between Zhenshan 97B and Nan Yang account for have been designed, for the Fine Mapping analysis of qpc1.The sequence information of these marks is in Table 1.

Table 1. is for the primer of map based cloning of the present invention and gene function analysis

3. the Fine Mapping of the analysis of restructuring individual plant and qpc1 and candidate gene are determined

For between the positioning area that further dwindles qpc1, from 10008 strains by BC ₃F ₁The BC that individual plant is derivative ₃F ₂Pick out the restructuring individual plant in large group.

At first with SSR mark RM472 and RM104 mark, screened, from 6000 individual plants, (colony 1) finds 52 restructuring individual plants altogether, and these individual plants are confirmed the phenotype of its previous generation through progeny testing.Further by the qpc1 assignment of genes gene mapping between PB2 and PB7, between two marks, respectively exist 4 the restructuring individual plants support this positioning result.From 4008 individual plants, (colony 2) screens respectively 5 and 3 restructuring individual plants with PB1 and these two primers of PB2, utilize Indel mark (PB9-PB12) newly developed, screen the restructuring individual plant in conjunction with two large groups, with the qpc1 assignment of genes gene mapping between PB11 and PB12 (Fig. 4) the most at last of overlapping graphing method.The physical extent (Fig. 4) of the approximately 19.8-kb that this interval lists corresponding to the genome sequence of Nipponbare and 93-11.Between PB11 and PB12, (by applicant oneself, designed, referring to table 1) find a complete full-length cDNA in the 19.8-kb scope that defines, be numbered AK121636 (Rice Genome Annotation Proiect Rice GenomeBrowser-Release 7).The cDNA sequence total length 1401bp of the present invention clone's qpc1 gene, with the gene of unique complete total length in the 19.8-kb scope, mate fully, so this cDNA is the unique reliable candidate gene of qpc1.

The transgenosis test of embodiment 3:qpc1

Real-Time PCR expression pattern analysis is found, this gene all has expression in each tissue in the time of infertility, and the kind (Zhenshan 97B) that protein content is high in clever shell endosperm is than the expression amount of protein content low (Nan Yang accounts for) high (Fig. 5), prediction is expressed and is measured vital role, so candidate gene full length cDNA sequence according to prediction, design the coding region sequence of the qpc1 gene of a pair of special primer cDNA (in Table the primer sequence of 1 last column) amplification Zhenshan 97B, then be connected on TA cloning vector (deriving from the business carrier of U.S. Promega company), pick out the correct clone without sudden change who contains candidate gene, carry out again subclone, be connected to double base overexpression vector pCAMBIA1301S and (see Fig. 9, this carrier is so kind as to give by Australian CAMBIA laboratory) on, in addition, use the same method the transgenosis of Zhenshan 97B promotor and coding region (ZpZc) is connected to binary expression vector pCAMBIA1301 (transformation on the basis of the double base overexpression vector pCAMBIA1301S that this carrier is so kind as to give by Australian CAMBIA laboratory) upper (seeing Figure 10).Design a pair of special primed RNA i (table 1) in qpc1 coding region 3 ' that is positioned at, by the coding region of its 580bp, forward and backward chaining suppress expression vector ds-pCAMBIA1301 (ds1301) upper (seeing Figure 11, transformation on the basis of the double base overexpression vector pCAMBIA1301S that this carrier is so kind as to give by Australian CAMBIA laboratory) to double base respectively at twice.Adopt transgenic method, the correct clone's that obtains plasmid is imported to the precious Shan 97 of rice varieties, Nan Yang accounts for and middlely spends 11 in (in spend 11 are commercial varieties that Institute of Crop Science, Chinese Academy of Agricultural Science cultivates) by agriculture bacillus mediated rice transformation system, through inducing, subculture, infect, cultivate altogether, callus that screening has hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain genetically modified paddy rice plantlet.Agriculture bacillus mediated japonica rice subspecies genetic conversion system is mainly applied the method for the people such as Hiei report (referring to Efficient transformation of rice, Oryza sativa L, mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, PlantJournal 6:271-2820) also be optimized on this basis.The genetic conversion system of agriculture bacillus mediated long-grained nonglutinous rice subspecies is mainly applied the method for the people such as Lin report (referring to Optimizing the tissue culture conditions for high efficiency transformation of indica rice.PlantCell Rep, 2005,23:540-547), detailed genetic transformation step is with reference to He Yuqing, the granted patent of Li Yibo (patent No. ZL201010188458.6, publication number: the CN101880671A) method of report operation.

The present invention obtains independent transgenosis Over-expression T altogether ₀Account for rice plant 51 strains for Nan Yang, comprise the positive individual plant of 22 strains and the negative individual plant of 29 strains, the T obtained ₁Take OX (NYZ)-55 family and OX (NYZ)-66 family for rice plant is example; Transgenosis Over-expression T ₀Dai Zhonghua 11 rice plant 71 strains, comprise the positive individual plant of 30 strains and the negative individual plant of 41 strains, the T obtained ₁Take OX (ZH11)-76 family and OX (ZH11)-77 family for rice plant is example; Precious Shan 97 promotors merge the independent transgenosis T in coding region (ZpZc (NYZ)) of self ₀For rice plant 63 strains, comprise the positive individual plant of 24 strains and the negative individual plant of 39 strains, the T obtained ₁Take ZpZc (NYZ)-18 family and ZpZc (NYZ)-30 family for rice plant is example; RNAi suppresses to express the T that transforms precious Shan 97 ₀For plant 59 strains, the T obtained ₁Take RNAi (ZS97)-44 family and RNAi (ZS97)-47 family for rice plant is example; RNAi suppresses to spend 11 T in the expression conversion ₀For plant totally 68 strains, comprise the positive individual plant of 31 strains and the negative individual plant of 37 strains, the T obtained ₁Take RNAi (ZH11)-7 family and RNAi (ZH11)-12 family for rice plant is example (Fig. 6 A, Fig. 6 B, Fig. 7).The protein content of these five kinds of transgenic paddy rices (OX (NYZ), OX (ZH11), ZpZc (NYZ), RNAi (ZS97), RNAi (ZH11)) positive and negative plant is at T ₀Generation and T ₁Variant significantly (P<0.001) (Fig. 6 A, Fig. 6 B) in generation.And at T ₁In generation, be divided into from detect finding, protein content and genotype be divided into from (Fig. 7), proved that this unique candidate gene is exactly qpc1 QTL.In addition, the transgenosis that the Zhenshan 97B promotor merges himself coding region is consistent with the above results, all can increase this phenotype of protein content (Fig. 6 A, Fig. 6 B, Fig. 7), complementary success, this QTL gene is successfully cloned, and illustrate that promoter region is the reason of qpc1 heritable variation, and explanation qpc1 is a positive regulatory factor of controlling protein content.Also proved that this gene can improve rice quality by genetic transformation simultaneously, for cultivating the High-quality Cultivation rice varieties, provide genetic resources.

(2) qpc1 function prediction

According to InterProScan (http://www.ebi.ac.uk/InterProScan/), protein structure is predicted, the protein of qpc1 genes encoding is comprised of 466 amino acid, comprise a conservative PF01490 structural domain, be predicted as the cross-film amino acid transporter, belong to the APC superfamily.By PROSITE (http://www.expasy.ch/prosite/) Analysis and Identification this albumen have a plurality of avtive spots (13 N-myristoylation sites; 1 amidation site; 2 N-glycosylations; 2 casein kinase i I phosphorylation sites; 4 protein kinase C phosphorylation sites, 1 cAMP and cGMP deopendent protein kinase phosphorylation site).Research in the past shows that this protein family all exists in animal, plant, yeast and bacterium, has the function of a kind of a, class of transhipment or multiple amino acids, and participates in the middle of regulate several biological processes.

Embodiment 4: relatively the natural variation between qpc1 allelotrope is determined in order-checking

(1) sequencing

Kind Zhenshan 97B and the protein content low kind Nan Yang high to endosperm protein content account for the order-checking that kind is carried out the target section.Utilize 15 PCR primers mutually to combine (in Table 2), adopt Hi-Fi LA-Taq and rTag (purchased from precious biotechnology Dalian company limited) to carry out pcr amplification from the genome of these two kinds, the sequencing kit then provided according to U.S. Perkin Elmer company (Big Dye Kit) carries out the PCR order-checking.Use Sequencher 4.5 softwares (U.S. Gene Codes Corporation) splicing sequence.The DNA sequence dna of the major gene qpc1 that the clone obtains (is cloned and is obtained) as shown in sequence table SEQ ID NO:1 from Zhenshan 97B, and the allelic DNA sequence dna of this major gene qpc1 is as shown in sequence table SEQ ID NO:2 (from Nan Yang accounts for, the clone obtains).

The primer that table 2 relatively checks order for the present invention

2. the sequence of natural variation occurs relatively

Between accounting for, the kind Zhenshan 97B that the intersegmental sequence comparing analysis in target area is high at endosperm protein content and the low kind Nan Yang of protein content carry out (Fig. 8), discovery the promotor of 5.9-kb and coding region scope internal protein content is high and low these two kinds between common SNP and the InDel differences of 23 of existence, wherein 15 sudden changes are positioned at promoter region, 7 sudden changes are positioned at the 1st intron of coding region, and 1 sudden change is positioned at the 4th exon (same sense mutation) and does not cause amino acid variation (Fig. 8).Account for (protein content is low) and can improve the protein content that Nan Yang accounts for because the promotor with precious Shan 97 (protein content is high) self and coding region sequence transform Nan Yang, can account in successful complementary Nan Yang, therefore the variation of qpc1 promoter region is the reason that causes genovariation, and then causes the variation of paddy endosperm protein.

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Claims (4)

1. the major gene qPc1 of the control paddy endosperm protein content proterties of a separating clone, its nucleotide sequence is as shown in sequence table SEQ ID NO:1.

2. the allelotrope of major gene claimed in claim 1, its nucleotide sequence is as shown in sequence table SEQ ID NO:2.

3. the application of major gene claimed in claim 1 in controlling paddy endosperm protein content proterties.

4. the application of the allelotrope of major gene claimed in claim 2 in controlling paddy endosperm protein content proterties.

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