Functional Study Of Candidate Genes For Rice Yellow Leaves

The normal development of chloroplast is critical for plant growth.The study of leaf color mutants is an effective way to elucidate gene function during chloroplast development.In recent years,good progress has been made in the study of genes related to leaf color mutation.The rice leaf color mutants that have been reported mainly include yellowing,albino,bright green,streak streaks,thermosensitive discoloration,greening and transgenic purple.In a previous study,a yellow leaf mutant was screened from the progeny of indica rice 9311 irradiated with ⁶⁰Co-?radiation.Using the yellow leaf mutant and wild type as materials,combined with resequencing,map-based cloning and transcriptome sequencing,13 differentially expressed genes were found in the region of mapped cloning.To further determine whether each of the above genes is associated with a yellow leaf mutation,the content and results of this study are as follows:1.A series of editing vectors for the rice yellow leaf mutant candidate genes were constructed using the CRISPR/Cas9 system and transformed.Based on the two target site sequences in each exon region,the pC1300-Cas9 editing vectors of 13 candidate genes were constructed,It was transferred to Nipponbare rice varieties by Agrobacterium-mediated transformation,while the gene editing vectors of BGIOSGA012976 and BGIOSGA010427 simultaneously transformed 9311 varieties.Except for the genetic transformation of the four genes BGIOSGA010427?acceptor9311?,BGIOSGA010449,BGIOSGA012911 and BGIOSGA012927,the T₀ generation positive transgenic plants were obtained.The editing rate statistics showed that the editing rate of T₀-Cas9-12976 transgenic plants was 18.5%,T₀-Cas9-10448 was 18%,T₀-Cas9-12931was 41.3%,T₀-Cas9-12963 was 15.4%,T₀-Cas9-12987 was 51.5%,T₀-Cas9-10427 and T₀-Cas9-10375 was 0.After transferring pC1300-Cas9-12976 to Nipponbare and 9311,the leaf color of T₀ generation transgenic editing seedlings was similar to that of yellow leaves mutant and was different that of Nipponbare and 93112.The interference expression vectors of yellow leaf candidate genes BGIOSGA012976 and BGIOSGA010427 were constructed by RNAi technology.Among the 13 differentially expressed genes,the BGIOSGA012976 and BGIOSGA010427 genes were predicted to belong to the family members of the magnesium ion chelatase I subunit,but their biological functions have not been reported yet.Compared with 9311 and yellow leaf mutant,SNP existed in both genes.The first and second chains of the target genes BGIOSGA012976 and BGIOSGA010427 were inserted into the target vector DS1301 by two digestions,respectively.The interference vectors of two yellow leaf candidate genes have been constructed successfully,After transformation of rice 9311,BGIOSGA010427obtained a positive transgenic plant with a greenish leaf color and a similar phenotype to 9311.Therefore,the correlation between BGIOSGA010427 gene and yellow leaf needs to be further determined.3.Overexpression vectors of yellow leaf mutant candidate genes BGIOSGA012976and BGIOSGA010427 were constructed by overexpression technology.Candidate genes BGIOSGA012976 and BGIOSGA010427 were analyzed by bioinformatics software.Specific primers were designed and overexpression vectors were constructed.Agrobacterium-mediated transformation of pCAMBIA1302-C10427 and PCAMBIA1302-C12976 has been completed,The genetic transformation of japonica rice is difficult,and the genetic transformation is currently in the differentiation stage.