Map-based Cloning And Functional Analysis Of QGPC-10,a QTL Responsible For Rice Grain Protein Content

Rice(Oryza sativa L.)is one of the most important food crops in China or even in the world In recent decades.the rice yield level in China has been continuously improved.However,the rice grain quality is generally lower than that of other countries.Chinese rice breeders have carried out many studies on the genetic analysis and improvement of rice grain quality,especially on the starch quality.Although the improvement of starch quality has achieved great success,the current status of rice grain quality still can not meet the growing demand of domestic consumers.Therefore,the improvement of rice grain quality only in the aspect of starch property is not enough,one of the important reasons is that the grain protein content(GPC),the second component of rice endosperm,is acting an important factor,seriously affecting rice grain quality,especially the nutritional quality and cooking and eating quality.Therefore,it is of great significance for rice quality improvement to analyze the genetic regulation mechanism of rice GPC and clone the major genes in control of GPC in rice.In this study,the GPC of different types of rice germplasm was determined to explore the genetic variation and the main factors causing rice GPC variation.At the same time,a set of chromosome single fragment substitution line(CSSL)derived from indica-japonica cross was used to conduct QTL mapping for rice GPC in multiple environments.Furthermore,the identified major QTL qGPC-10 was cloned and functional analysized.The main research results are as follows:1.The genetic variation and distribution of rice grain protein contentThe investigation of the GPC of 402 rice germplasm resources in different environments showed that there is a wide range of continuous variation of the GPC in the natural population,showing a single peak normal distribution with a range of about 8%.The average GPC varies by nearly 2%between years,indicating that environmental conditions have a great influence on it Further analysis showed that there were more varieties with low GPC in japonica subspecies and more varieties with high GPC in indica subspecies.The analysis of the contents of four storage proteins in 103 indica/japonica varieties with similar heading date and plant height showed that the variation of glutelin content in population could explain 73.9%of the variation of total protein content,indicating that glutelin content was the main factor for the difference of GPC in indica/japonica rice2.Fine mapping of rice grain protein content QTL qGPC-10In order to further explore the genetic regulation mechanism of rice GPC,a set of chromosome single segment substitution lines(CSSL)derived from Sasanishiki and Habataki hybrid was used to determine the GPC in five different environments.The results showed that the GPC of indica rice variety Habataki was significantly higher than that of japonica rice Sasanishiki,and the GPC was continuously and widely distributed in CSSL population,indicating that the rice GPC was a typical quantitative trait.The results of QTL mapping showed that 13 QTL loci were identified in 5 environments,among which qGPC-1 and qGPC-10 could be detected repeatedly in 5 environments.The results indicated that qGPC-1 and qGPC-10 may not be sensitive to the environment and are the key sites causing the difference in protein content of rice in this population.The qGPC-10 was limited to about 35Kb on chromosome 10 by using high generation population derived from the hybridation between CSSL SL431 and its parent Sasanishiki,in which there were four ORFs.Comparison of the expression of the four ORFs in different tissues of NILs showed that only LOC_Os10g26060 was significantly different in the endosperm of NILs(P=0.0003).3.The cloning and functional analysis of major QTL qGPC-10Gene knockout and complementary transgenic analysis showed that LOC_Os10g26060 was a candidate gene for qGPC-10 encoding a glutelin precursor protein,OsGluA2.RT-PCR analysis showed that OsGluA2 was specifically expressed in the endosperm,and the expression level of indica allele was significantly higher than that of japonica.The results of gene sequencing showed that there were 5 haplotypes of OsGluA2 in the natural population,among which the expression level and glutelin content of type 1 were significantly lower than those of the other 4 types.Fusion expression analysis showed that a SNP(-1100)located in the OsGluA2 promoter region was correlated with its transcriptional expression level,and the allele of OsGluA2 could be divided into two alleles:low expression(OsGluA2LET)and high expression(OsGluA2HET).Population genetic and evolutionary analysis showed that OsGluA2 was not artificially selected during rice domestication,and OsGluA2LET.which was mainly distributed in japonica rice,originated from wild rice.The analysis of NIL and transgenic rice storage materials and the physicochemical characteristics of starch showed that OsGluA2 was a positive regulator of the GPC of rice,and the higher the expression level,the higher the content of glutelin,and the larger the volume of protein body ? deposited with glutelin.In addition,the gene also affected the expression of starch-related qualities and the contents of amino acids in rice,which had multiple effects on rice quality.