CN107937409B - Clone and application of rice tillering angle gene TAC3 - Google Patents
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Abstract
The present invention belongs to the field of plant gene engineering technology. In particular relates to the cloning and the application of a rice tillering angle gene TAC 3. The sequence of the TAC3 gene is shown as SEQ ID NO: 1, the cDNA sequence is shown as SEQ ID NO: 2, the sequence of the encoded protein is shown as SEQ ID NO: 3 and 4. The QTL locus related to the rice tillering angle is positioned by GWAS, the range of qTA3 is narrowed by combining LD analysis, and a candidate gene TAC3 is determined by combining a reverse genetics procedure. The function of TAC3 gene is verified through a function-acquired mutant TAC3D-1, the expression level of the gene in TAC3D-1 is obviously improved, and compared with wild ZH11, the tillering angle at the tillering stage is obviously improved from 10.4 degrees to 19.4 degrees, and the tillering angle at the flowering stage is obviously improved from 8.4 degrees to 17.6 degrees. The TAC3 gene has obvious difference in tiller angle in different haplotype indica rice materials.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering. In particular to the cloning and the application of a rice tillering angle gene TAC3, wherein the gene is positioned on a third chromosome of rice and controls the tillering angle of a rice plant.
Background
The tillering angle of rice is one of the main characters for forming an ideal plant type, and determines the planting density per unit area and the crop yield of plants. The ideal tillering angle can avoid diseases induced by high humidity due to over-small angle, and can also avoid reduction of photosynthesis efficiency and yield per unit area due to creeping growth, so the ideal tillering angle is selected by human beings in the long-term domestication and genetic improvement processes of rice. However, studies on the genetic molecular mechanism of rice tillering angle have not progressed rapidly until the last 30 years.
At the end of the last century, researchers have used classical parental hybridization mapping to explore a number of QTL (quantitative trail Loci) sites that control rice tillering angles (Li et al, 2006, New phytologist 170: 185-. In The last 10 years, tillering angle related genes such as LAZY1, TAC1, PROG1, LPA1, SOLs and The like (Li et al, 2007, Cell research 17: 402-.
The tillering angle of rice has large natural variation among different varieties, however, the existing genes for controlling the tillering angle are very limited, especially the related genes for controlling the natural variation of the tillering angle. Therefore, at present, more genes related to tillering angles need to be excavated to better perfect the genetic basis of the genes, so as to assist breeding. In recent years, genome-wide association analysis (GWAS) has been widely introduced in crops as an effective method for detecting natural variation of complex traits (Huang and Han, 2014, Annual review of plant biology 65:531 551), which makes full use of abundant phenotypic variation and genetic variation among crop varieties based on a large number of DNA markers and associations among phenotypic traits (Rafalski, 2010, Current opinion in plant biology 13: 174-180).
So far, no report about the application of the TAC3 gene in the regulation of the rice tillering angle is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, a gene which is positioned on a third chromosome and used for controlling the tillering Angle is separated and cloned from rice, the applicant names the gene as TAC3(Tiller Angle Control 3) gene, and the tillering Angle of the rice is improved by utilizing the gene, so that the aim of controlling the plant type and the yield of the rice is fulfilled.
The technical scheme of the invention is as follows:
the TAC3 gene related by The invention is based on SNPs (refer to Zhao et al, 2015, Nucleic acids research 43: D1018-D1022) of 529 cultivated rice (Oryza sativa) existing in national center laboratory of China university of agriculture crop genetic improvement and corresponding flowering phase tillering angle phenotype data for GWAS analysis, and is further combined with mutant 05Z11AZ62 (The mutant information is published in rice mutant library of national center laboratory of China university of agriculture crop genetic improvement, downloading website: http:// rmd.ncr.cn/. reference: Wu et al, 2003, The Plant Journal 35: 418. 427; Zhang et al, 2007, The Plant Journal 49: 947. 959), cloned gene controlling rice tillering angle on a third chromosome, and a TAC coding putative protein of The TAC gene, wherein The TAC coding protein is used for controlling rice at 3. The separation and cloning of the TAC3 gene are helpful for understanding the genetic mechanism of rice tillering angle, and provide new gene resources for cultivating ideal plant types.
The application of a separated TAC3 gene in controlling the tillering angle of rice, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.
The application of a separated TAC3 gene in controlling the tillering angle of rice, wherein the protein sequence coded by the gene is shown as SEQ ID NO: 3, respectively.
The application of a separated TAC3 gene in controlling the tillering angle of rice, wherein the protein sequence coded by the gene is shown as SEQ ID NO: 4, respectively.
The nucleotide sequence and the protein sequence of the TAC3 gene can be further applied to rice plant type improvement and rice breeding.
The more detailed technical scheme is as follows:
the invention utilizes a whole genome association analysis (GWAS) method to position the QTL locus related to the rice tillering angle, further combines Linkage Disequilibrium (LD) analysis to narrow the QTL range, and finally combines a reverse genetics section to determine candidate genes.
The group of the invention for GWAS analysis has reported the application of other multiple traits (such as metabolism, heading stage, spike type and the like) besides the application of controlling the rice tillering angle trait (Chen et al, 2014, Nature genetics 46: 714-721; Yang et al, 2014, Nature communications 44: 92-96).
According to the invention, 295 parts of indica rice material is utilized to position qTA3 on a third chromosome 29,504,013-29,791,496 physical position (MSU.V6), LD analysis is further carried out to reduce the number of genes to 2 as candidate genes, and finally LOC _ Os03g51660, namely TAC3, is determined as the candidate gene of the site through separation identification, gene expression profiling analysis and haplotype analysis by screening the mutant of the invention. The mutant 05Z11AZ62 is a mutant with a function of TAC3 gene, the mutant 05Z11AZ62 with a new function is named as TAC3D-1, the expression level of the TAC3 gene in TAC3D-1 is obviously improved, and the corresponding tillering angle at the tillering stage is obviously improved to 19.4 degrees from 10.4 degrees and is obviously improved to 17.6 degrees from 8.4 degrees compared with the wild type middle flower 11(ZH 11). The 295 parts of indica rice material TAC3 have obvious difference in tiller angle among different haplotypes, which provides a molecular basis for later genetic breeding.
Compared with the prior art, the invention has the following outstanding advantages and effects:
(1) the invention reports a gene TAC3 for regulating the tillering angle of rice for the first time, and provides a new gene resource and a genetic basis for cultivating an ideal plant type of rice.
(2) The invention combines GWAS, LD, gene expression profile analysis and mutant coseparation identification methods, quickly and efficiently clones genes, and provides technical reference for research of related trait genetic mechanisms and gene cloning in other crops.
Drawings
SEQ ID NO: 1 is the DNA sequence of TAC3 gene, and the sequence total length is 1717 bp.
SEQ ID NO: 2 is a cDNA sequence of the TAC3 gene, the present invention relates to two transcripts whose transcription start sites are different, and therefore, in this sequence, the base sequence at positions 1 to 408 is transcript 1, namely coding region 1(CDS 1); the nucleotide sequence from 56 to 169 is transcript 2, coding region 2(CDS 2).
SEQ ID NO: 3 is a protein sequence coded by CDS1 of TAC3 gene and codes for a protein sequence of 135 amino acids.
SEQ ID NO: 4 is a protein sequence coded by CDS2 of TAC3 gene and codes for a protein sequence with 37 amino acids.
FIG. 1: and 295 parts of GWAS analysis result of the tiller angle of the indica rice in the flowering period. Description of reference numerals:
the graph a in fig. 1 is (left graph) manhattan and (right graph) quantile-quantile graphs obtained by GWAS analysis through a linear regression model (LR) according to 295 parts of tiller angle data of indica rice at the flowering stage in Hainan China; the b picture in figure 1 is the graph of manhattan (left picture) and quantile-quantile (right picture) obtained by performing GWAS analysis by LR according to the data of the tillering angle at the flowering stage of 295 parts of indica rice in Wuhan China.
FIG. 2: LD analysis of the TAC3 candidate gene. The reference mark indicates that r between every two polymorphic Sites (SNP) in two candidate genes is calculated2Value (measure of LD value), the magnitude of which is indicated by the scale. Black bold line indicates lead SNP position.
FIG. 3: statistical analysis results of the tac3D-1 mutant phenotype picture and the tillering angle. Description of reference numerals:
the a picture in figure 3 is a picture of the phenotype of tac3D-1 mutant at the tillering stage; FIG. 3, panel b is a photograph of the phenotype of tac3D-1 mutant at flowering stage; the c picture in figure 3 is the statistical analysis result of tillering angle of the Wuhan summer tac3D-1 mutant in the China in 2015 and 2016. In fig. 3: WT, for wild type ZH 11; tac3D-1H, representing a heterozygous mutant; tac3D-1M, indicating homozygous mutant; the obvious difference of the tillering angle between the mutant and the wild type is shown, p is less than 0.001; the obvious difference of the tillering angle between the mutant and the wild type is shown, and p is less than 0.01. FIG. 4: the result of coseparation identification of tac3D-1 mutant. Description of reference numerals:
panel a in FIG. 4 is the cosegregation identification at the DNA level. L and R respectively represent left and right primers which are positioned on the rice genome and used for identifying the mutant, and N represents a primer on a carrier. Panel b in FIG. 4 is a representation of co-segregation characterization of expression levels. W and WT, both wild-type ZH 11; h and tac3D-1H, both representing a heterozygote mutant; m and tac3D-1M, both representing homozygous mutants; it shows that compared with wild type, the expression level of TAC3 gene has significant difference, p is less than 0.001.
FIG. 5: and (3) analyzing a space-time expression profile of the TAC3 gene in the whole growth period of the rice. Description of reference numerals:
the used material is wild rice variety Zhonghua 11(ZH 11); the symbol 1 indicates a tissue sample at the tillering stage, and the symbol 2 indicates a tissue sample at the flowering stage.
Detailed Description
Example 1: GWAS analysis of tillering angle in rice flowering period
1. Investigation of tillering angle in rice flowering phase
The investigation object is 529 parts of Asian cultivated rice germplasm resources including local varieties and fine varieties, and the resources are divided into 9 small subgroups: indI, indII, indica intermediate, Tej, Trj, japonicuma intermediate, Aus, VI and intermediate. Among them, 3 small subgroups of indI, indiii and indica intermediate types belong to the indica sub-group consisting of 295 varieties (Zhao et al, 2015, Nucleic acids research 43: D1018-D1022). The population has rich phenotypic variation, and phenotypic variation and genetic basis of natural population of rice are comprehensively analyzed by utilizing the phenotypic variation and GWAS genetic analysis of metabolic traits and agronomic traits such as flowering phase, Plant height, spike type and the like (Chen et al, 2014, Nature genetics 46: 714-721; Yang et al, 2014, Nature communications 44: 92-96; Han et al, 2016, front. Plant Sci.7: 1270; Bai et al, 2016, Plant Genome). The tillering angle of the rice is investigated about 5 days after the rice blooms, the angle between two tillers with the farthest distance is investigated by using a protractor, and half of the numerical value is the tillering angle of the single plant. The population was respectively surveyed in the winter of 2013 in Hainan China and in the summer of 2014 in Wuhan China.
2. GWAS analysis of tillering Angle
Illumi for 529 parts of Asian cultivated ricena Genome Analyzer II sequencing analysis, overall genomic DNA sequence coverage was approximately 2.5-fold for genotyping (Chen et al, 2014, Nature genetics 46: 714-721). In the FaST-LMM program, 3916415, 2767159 and 1857845 SNPs were respectively contained in the total population and the indica rice subpopulation while satisfying the conditions of MAF (minor alloy frequency) 0.05 or more and the minor alloy variety number not less than 6 and used for GWAS analysis by two Methods, LMM (linear texture method) and LR (linear regression) (Lippert et al, 2011, Nature Methods 8: 833-. Group structure and kinship (kinship) are considered as auxiliary considerations when GWAS analysis is performed by LMM methods. 757578, 571843 and 24348 potent independent SNPs were obtained in the total population and indica rice subpopulation, respectively (Li et al, 2009, Bioinformatics 25: 2078-2079). P-value was 1.3X 10 in the total population and the indica rice subpopulation, respectively-6、1.8×10-6And 4.1X 10-6As an effective threshold for significant associated signals detected simultaneously in south of Hainan, China and Wuhan, China using the LMM method; since LMM leads to a reduction in the number of sites for false negatives and LR leads to the appearance of false positive sites, the p value is 1.0X 10-8A valid threshold for the associated signal is detected as the LR method and the first 5 that satisfy this condition are selected for subsequent study. qTA3/TAC3 in the invention is a significant association site detected by GWAS analysis by LR method in indica rice subgroup, and p values of lead SNP (both sf0329582676) of data of Hainan and Wuhan in China are 3.9 × 10 respectively-16And 1.1X 10-23(FIG. 1).
Example 2: qTA3 determination of candidate genes for association sites
1. Linkage Disequilibrium (LD) analysis
Linkage disequilibrium was determined by the TASSEL5.0 software using the normalized coefficient of disequilibrium (D') and the square of the allele type coefficient between the paired SNP sites (r)2) And (5) carrying out measurement study. The mean LD recession values of the study populations of the present invention have been reported genome-wide, 167kb, 93kb and 171kb in the total population and indica subpopulation, respectively (Xie et al, 2015, Proceedings of the National Academy of Sciences of the United States of America 112: 5411-.We calculated the LD decay interval covering lead SNPs as follows: first, r between the lead SNP and all SNPs in the upstream and downstream 2Mb regions of the lead SNP is calculated2A value; then using lead SNP and r of the first 10% in the interval of 1.5Mb to 2Mb of the upstream and downstream2The average of the values was taken as background; r calculated in the last step2The continuous interval with the value minus the background value larger than 0.2 is the attenuation interval of lead SNP. The LD attenuation interval of qTA3/TAC3 is 29504013-29791496. Further analysis by the Haploview4.2 software gave r between all SNPs between LOC _ Os03g51670 and LOC _ Os03g51660 containing lead SNP (sf0329582676)2Values in which some SNPs show high linkage disequilibrium, the 5' end of LOC _ Os03g51660 is located within one LD cell with LOC _ Os03g51670 (FIG. 2). The above results suggest that our LOC _ Os03g51660 and LOC _ Os03g51670 may be candidate genes for qTA3/TAC 3.
Co-isolation characterization of tac3D-1 mutants
In order to rapidly identify candidate genes, mutants of LOC _ Os03g51660 and LOC _ Os03g51670 genes with T-DNA insertion mutation were found in rice mutant information published by RiceGE database (http:// signal.salk. edu/cgi-bin/RiceGE), respectively. Wherein The mutant 05Z11AZ62 is a Zhonghua 11(ZH11) background, and is derived from a mutant library of a national key laboratory of The university of agriculture of Huazhong crop genetic improvement (mutant library website: http:// rmd. ncpgr. cn/. reference is Wu et al, 2003, The Plant Journal 35: 418-427; Zhang et al, 2007, The Plant Journal 49:947-959), and The T-DNA insertion site is a promoter region 991bp away from The LOC _ Os03g51660 start codon. The mutant is planted in the experimental field of agriculture university in Wuhan Huazhong China in 2015 (60 individuals) and 2016 (96 individuals) respectively, the tillering angle of the mutant at the tillering stage and the flowering stage is investigated, and the isolated individual with the tillering angle increased in two-year repeated planting is statistically observed (a picture in figure 3 and b picture in figure 3).
In order to prove whether the individual plant with the tiller angle remarkably increased compared with the wild type is caused by T-DNA insertion, fresh leaves of all the individual plants of the mutant family are selected to extract DNA, and whether the gene and the phenotype are co-separated is proved on the DNA level. For DNA extraction, the CTAB method was referred to (Zhang et al, 1992, Theor Appl Genet,83, 495-499). PCR amplification is carried out from genomes of all single plants of the mutant family by using 2 pairs of PCR primers (L1+ R1, N1+ R1 and 3 primers in total, sequence information of which is provided by a RiceGE database, L1 and R1 primers are primers on rice genomes, N1 is a carrier primer, sequences of which are L1-CGAGTAGCTACGGATGAGGC, R1-TTCTTCAACTCTGATGGGGC and N1-AATCCAGATCCCCCGAATTA) and R-Taq (the total PCR reaction is 20 mu L, specifically, 2 mu L of DNA first chain template, 10xPCR buffer 2 mu L, 10mM dNTP 1.5 mu L, 0.3 mu L of each bidirectional primer, 0.2 mu L of R-Taq enzyme, and double distilled water are added to 20 mu L. the PCR buffer, dNTP, R-enzyme and the like which are purchased from BAO bioengineering Co Ltd. PCR reaction conditions are that PCR reaction is carried out at 4 minutes at 94 ℃, 30 seconds at 30 seconds, 60 seconds at 60 ℃ and 58 ℃ from 35-58 rd step, sixthly, storing at 72 ℃ for 7 minutes and at 4 ℃). The PCR products were checked by agarose gel electrophoresis, and the individuals corresponding to the bands amplified by the primer combination L1+ R1 were wild-type plants without T-DNA insertion, the individuals corresponding to the bands amplified by the primer combination N1+ R1 were homozygous mutant plants with T-DNA insertion and indicated by M, and the individuals corresponding to the bands amplified by the primer combinations of both pairs were heterozygous mutant plants with T-DNA insertion and indicated by H (FIG. 4 a). Counting the tillering angles of the 3 genotypes of the single plants respectively, and performing t-test detection (fig. 3c), the results show that the average value of homozygous mutant M tillering angles at the tillering stage and the flowering stage of two years is the largest, the average value of heterozygous mutant H tillering angles is centered, the wild type W is the smallest, and significant differences exist between the mutant and the wild type, so the mutant is named tac3D-1, and the specific results are as shown in table 1 below:
TABLE 1 statistics of tillering angle of tac3D-1 mutant family at tillering stage and flowering stage
P < 0.001; p <0.01
In order to prove whether the mutant is a function-acquiring mutant, namely whether the tillering angle of a mutant plant is increased due to the increase of the expression level of LOC _ Os03g51660, in 2016 summer, a leaf blade which just begins to generate a tillering period is selected to extract RNA, and each genotype selects not less than 9 plants for expression level detection. The cDNA sequence was amplified by a conventional RT-PCR method (see: J. SammBruk, EF Frizi, T Mannich, Huangpetang, Wangjia seal, et al, molecular cloning instructions (third edition), Beijing, scientific Press, 2002). The specific amplification method of cDNA is as follows:
1) firstly, extracting RNA of leaves at the tillering stage at the beginning, wherein a Trizol extraction kit (the specific operation steps are shown in a kit specification) of Invitrogen company is used for RNA extraction;
2) reverse transcription in RT-PCR to synthesize the first strand of cDNA: mixing the mixed liquid 1: 4 μ g of total RNA, DNAse I2U, 10 × DNAse I buffer1 μ l, adding DEPC (diethylpyrocarbonate, a strong inhibitor of rnase) to treat water (0.01% DEPC) to 10 μ l, mixing, standing mixture 1 at 37 ℃ for 20 minutes to remove DNA, after 20 minutes, standing mixture 1 in a 65 ℃ water bath for 10 minutes to remove DNAse I activity, then on ice for 5 minutes, adding 1 μ l of 500 μ g/ml oligdT to mixture 1, iv immediately standing mixture 1 cooled on ice in a 65 ℃ water bath for 10 minutes to completely denature RNA, then on ice for 5 minutes, mixing mixture 2: mixing the mixed solution with 110 mu l of mixed solution, 4 mu l of 5x first strand buffer, 2 mu l of 0.1M DTT (mercaptoethanol), 1.5 mu l of 10mM dNTP mix, 0.5 mu l of DEPC treated water and 2 mu l of reverse transcriptase, putting the mixed solution 2 into a water bath kettle at 42 ℃ for warm bath for 1.5 hours after uniformly mixing, putting the mixed solution 2 into a dry bath at 90 ℃ for 3 minutes after the reaction is finished, and preserving the final reaction product at the temperature of-20 ℃. All reagents used in the reaction were purchased from Invitrogen;
3) then, specific primers are designed to amplify specific fragments by PCR according to the full-length cDNA sequence of the gene published by TIGR database (http:// rice. plant biology. msu. edu /). The specific qRT-PCR primer sequences of LOC _ Os03g51660 gene are as follows:
L2-CTTTGCTCCTCATCGCTGCT
R2-AGGCTCCTTGATCTGGTGATG
the specific qRT-PCR primer sequence of the UBQ serving as an internal reference gene is as follows:
L-AACCAGCTGAGGCCCAAGA
R-ACGATTGATTTAACCAGTCCATGA
4) and detecting the expression level of LOC _ Os03g51660 by a real-time fluorescent quantitative PCR method. Reagents were Roche (Roche, Mannheim, Germany) in a total reaction system of 10. mu.l, cDNA2.5. mu.l, 0.25. mu.M gene-specific primers, 5. mu.l Fast Start Universal SYBR Green Master (Rox) superMIX. The PCR instrument is QuantStaudio (TM)6Flex System, the PCR parameters are pre-denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 10 seconds after entering the cycle, annealing and extension at 60 ℃ for 40 seconds, and 45 cycles. As a result, as shown in the b-diagram in FIG. 4, the expression level of LOC _ Os03g51660 was significantly increased in the heterozygous mutant (tac3D-1H) and the homozygous mutant (tac3D-1M) compared to the wild-type control (ZH11W), and the average value of the gene expression level was higher in the homozygous single strain than in the heterozygosity.
The results show that the tillering angle of the TAC3D-1 mutant is increased in relation to the insertion of T-DNA into the LOC _ Os03g51660 gene promoter, and the tillering angle is in one-to-one correspondence with the improvement of the gene expression level, so that the TAC3 gene corresponding to qTA3 locus is LOC _ Os03g51660 and encodes a conservative hypothetical protein.
Analysis of TAC3 gene space-time expression profile in rice whole growth period
According to The existing research reports, genes for controlling The tillering angle of rice are specifically expressed at The tillering base part, such as TAC1(Yu et al, 2007, The Plant Journal 52:891-898), so that The expression profile analysis of The TAC3 gene can assist in proving The function of controlling The tillering angle and is helpful for understanding The molecular mechanism, thereby achieving The aim of assisting breeding. Tissue sites for gene expression profiling according to the invention include: the stem tip (length of 1-1.5cm) of a newly germinated rice plant, the tillering base, internodes, leaves, leaf sheaths and roots in the tillering stage, and internodes, leaves and leaf sheaths in the flowering stage. The expression level of TAC3 was detected by real-time fluorescent quantitative PCR, and the specific procedure was the same as described in the co-isolation identification of TAC3D-1 mutant of section 2 of example 2 to verify whether TAC3D-1 mutant is a gain-of-function mutant. The specific detection result is shown in figure 5, the expression level of the gene in each tissue of rice is not high, but the expression at the tiller base is obviously higher than that of other tissue parts, which also shows the regulation and control effect of the gene on rice tillering.
Example 3: haplotype analysis of TAC3 Gene
In order to better understand the natural variation of the TAC3 gene and achieve the aim of assisted breeding, 13 SNPs existing in 295 indica rice varieties of the TAC3 gene are extracted according to the SNPs information provided by a RiceVarMap database (http:// riceVarmap. ncpgr. cn /), 10 SNPs meeting the condition that the MAF is more than or equal to 0.05 are selected to construct haplotypes, and multiple comparisons are performed between main haplotypes (namely, the haplotypes meeting the condition that at least 10 varieties are contained) by using a Duncan test, and the results are shown in the following table 2:
table 2 multiple comparison of TAC3 to control tiller angle between major haplotypes in 295 parts of indica
Description of table 2: duncan test, p < 0.01.
As can be seen from the results in Table 2, the indica rice varieties with TAC3-Hap2 (such as rice variety "Yuexiangzhan") and TAC3-Hap3 (such as rice variety "9311", approved variety named Yangyao No. 6) have smaller tiller angles than the TAC3-Hap1 genotype, which is beneficial to close planting and full utilization of illumination. In the breeding process, indica rice varieties with TAC3-Hap2 and TAC3-Hap3 genotypes can be selected to be polymerized with other excellent characters, so that excellent varieties meeting more requirements of people are cultivated.
Claims (1)
1. The application of the separated TAC3 gene in controlling the tillering angle of rice is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.
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