EQTL Analysis Of Rubisco Activase Genes In Maize And Rice

Ribisco-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a key enzyme in plant photosynthesis and is directly involved in the CO2 fixation during carbon assimilation.In plants,Rubisco relies on the activation of Rubisco Activase(RCA)to function.A number of studies have shown that there is a significant positive correlation between RCA gene expression and plant photosynthetic rate and yield traits.Mapping the quantitative trait loci of RCA gene expression(eQTL)not only helps to understand the genetic mechanism regulating RCA gene expression,but also provides reference for high light efficiency and high-yielding crops.Maize and rice are important food crops in the world.Increasing the utilization of light energy of maize and rice plays an important role in further increasing the yield of both.Maize belongs to the C4 plant and rice belongs to the C3 plant.Compared to C3 plants,C4 plants show higher light energy utilization and biomass yield due to the extra CO2 enrichment mechanism.For a long time,people are interested in transforming C4 plants CO2 enrichment mechanism-related genes into C3 plants,such as PEPC gene,PPDK genes,etc.,to improve the photosynthetic efficiency of C3 plants.Few studies focus on the C3-pathway related photosynthetic genes that function in both C3 and C4 type plant,such as RCA gene and GAPDH gene.Whether the presence of CO2 enrichment mechanism of C4 plant maize changes its RCA gene expression intensity compared to C3 plant rice?Whether the interchanging of RCA gene expression regulatory system between maize and rice increase the RCA expression of one of these two species?These questions are not clear.In this study,the natural populations of maize and rice were used as materials to conduct the eQTL mapping of the maize RCA gene ZmRCAβ and rice RCA gene OsRCA;target DNA segment sequencing and candidate gene association were employed to screen for the elite alleles of promoter of the RCA gene in maize and rice;and callus genetic transformation and protoplast transient expression analysis were used to examine the differences of RCA gene promoter activity between maize and rice.The main findings are as follows:1.A total of 182 maize inbred lines genotyped with 558629 SNP markers were used as materials to determine the expression level of ZmRCAβ gene.Genome-wide association analysis(GWAS)detected 19 SNP markers significantly associated with ZmRCAβ expression level.Among them,the marker PZE-104005992 is located on the same chromosome as ZmRCAβ and is 3.6 Mb away from it,suggesting that this eQTL might be a cis-eQTL,while the rest might be regarded as trans-eQTL due to their longer physical distance away from ZmRCAβ.ZmRCAβ gene promoter sequence candidate association analysis detected two significant polymorphic loci associated with ZmRCAβ gene expression.Haplotype analysis divided ZmRCAβ promoter into three haplotypes,and cluster analysis further classified these haplotypes into two groups:Zm-GroupA and Zm-GroupB.The expression of ZmRCAβ in maize inbred lines with Zm-GroupA promoter was significantly higher than that of Zm-GroupB promoter.2.GWAS using 114 rice cultivars and 36,900 SNP markers detected eight SNPs significantly associated with OsRCA expression levels.Among them,the marker idl 1011480 is located on the same chromosome as the OsRCA and is 0.7Mb away from it,suggesting that this eQTL might be a cis-eQTL,while the rest might be regarded as trans-eQTL due to their longer physical distance away from OsRCA.Candidate association analysis of the OsRCA gene promoter sequence revealed that 12 polymorphic loci were significantly associated with OsRCA expression,two of which were located in the CAAT-box and the light-responsive element Box 4,respectively.Haplotype analysis divided the OsRCA promoters into five haplotypes,and cluster analysis further divided these haplotypes into two groups:Os-GroupA and Os-GroupB.The expression of OsRCA in rice cultivars with Os-GroupA promoter was significantly higher than that of Os-GroupB promoter.3.GUS expression vector and luciferase expression vector of four types of RCA promoters Zm-GroupA,Zm-GroupB,Os-GroupA and Os-GroupB were constructed,respectively.Genetic transformation using rice callus and transient expression analysis using maize and rice protoplast revealed that the Zm-GroupA promoter activity in maize was higher than that in Zm-GroupB genotype;the Os-GroupA promoter activity in rice was higher than that in Os-GroupB;and the rice RCA promoter activity was higher than that of maize RCA promoter.