Analysis And Evaluation Of Transgenic Rice Overexpression Of The RLRH Gene

Rice is one of the most important food crops in the world.With the continuous improvement of people’s living standards,people pay more attention to rice quality.Lysine is the first limiting essential amino acids for humans,but the lysine content is particularly lacking in rice,which leads to a serious effect on the nutrition utilization of rice.Therefore,promoting the lysine content of rice and improving its nutritional quality is a key problem that needs to be solved as soon as possible.Our research team has successfully identified two rice endogenous histone-like encoding genes RLRH1 and RLRH2 in previous studies,whose protein products were rich in lysine(>10 mol%).Moreover,the two genes were overexpressed in the elite japonica rice cultivar 9983.Preliminary analysis of the total lysine content of the transgenic rice from different constructions of RLRH1 and RLRH2 showed that the pGtl:RHRL1 and pGtl:mRLRH2 transgenic rice had the largest increment of total lysine content.Based on these previous results,the present study further screened the two constructs related transgenic rice to select more homozygous lines,and analyzed the expression of target genes and their total lysine content in rice grains.Moreover,three representative transgenic lines were selected from each construct to analyze their agronomic traits,rice quality and germination characteristics.In addition,RNA-seq analysis was performed to explore the possible molecular mechanisms of RLRH1 and RLRH2 overexpression on rice yield and quality,thus providing an important work basis for breeding and evaluating the high-lysine rice.The main research results are summarized as follows:1.A number of homozygous transgenic rice lines of pGt1:RLRHl and pGt1:mRLRH2 were successfully obtained.Moreover,the expression of target genes and the lysine contents were significantly increased in these transgenic lines.2.In terms of agronomic traits,the RLRH1 and RLRH2 transgenic lines had shorter and wider flag leaves,slender grains,decreased 1000-grain weight,and severe grain chalkiness.Moreover,plant heights of the transgenic lines were slightly reduced due to the notable shortened lengths of the last two internodes3.As to rice quality,the total lysine contents of the two transgenic lines were remarkably increased.Meanwhile,their total starch contents were decreased,and the soluble sugar and sucrose contents were increased.Moreover,the endosperm amylose content(AC)and gel consistency(GC)of the transgenic rice were reduced,and the gelatinization temperature was slightly increased.In addition,the starch viscosity of the transgenic rice was also elevated,therefore their overall taste values were improved.Furthermore,the digestion rate of the transgenic rice was faster than that of the wild type control.Further analysis of the starch fine structure showed that the distribution of chains with the degree of polymerization(DP)from 20 to 30 was notably increased while that from 30 to 50 was slightly reduced in the two transgenic rice when compared with that of the wild-type control.4.RNA-seq data showed that differentially expressed genes in the seeds of transgenic rice were involved in cell development and composition,biological regulation,metabolite regulation,enzyme activity regulation,etc.,indicating that RLRH1 and RLRH2 also modulated plant biological processes in addition to affecting rice lysine content.This is consistent with the fact that RLRH1 and RLRH2 function as histone analogs that may be widely involved in the regulation of gene expression.The RNA-seq results of some key genes were verified by qRT-PCR.Two representative genes(Os03g0377600 and Os10g0550100)were selected to generate their knockout mutants.Then their homozygous transgenic lines were selected and analyzed.The result indicated that the two transgenic rice had consistent field performance and grain quality changes as and RLRH2 lines,suggesting these two genes at least partially mediate the regulation of plant growth and seed development by RLRH1 and RLRH2.5.The pre-harvest sprouting phenotypes of high lysine rice was lighter than that of the wild-type control.The sprouting rate and speed of high lysine rice were reduced.The result of seed germination analysis was consistent with that of the pre-harvest sprouting assay.Further analysis indicated that the expression of ABA biosynthesis genes was increased while that of the ABA metabolic genes was decreased in the high lysine transgenic grains,which enhanced the accumulation of ABA in rice grains.We speculated that it may be one of the major reasons that result in decreased pre-harvest sprouting rates of the high lysine rice.