Functional Analysis Of Pistil Development Related Gene ST1 In Rice

IIRice yield and quality are closely related with pistil development.In the previous study,we found a novel XHS protein ST1,containing XH,Zf-XS,XS and Coiled-Coil domains,involved in pistil development,but its molecular mechanism is still unclear.We plan to research the function of ST1 on rice pistil development through identification of genotype and phenotype of mutant st1 and its complementary plants,functional analysis of its interacting protein and screening of regulated genes.The main progresses are as follows:(1)To study the function of each domain in protein ST1,the complement vectors AK3 containing Zf-XS and coiled-coil domains,AK5 including XH domain and AK6 containing Zf-XS domain were constructed and transformed into st1 homozygous callus,separately,and the progress is at differentiation stage.Meanwhile,the complementary plants AK1,AK2 and AK4 obtained from previous research were used for positive screening,expression level analysis and phenotype identification.The results showed the transcriptional levels of targeted sequences were significantly increased in all positive complementary plants compared to mutant st1,but still were lower than those in wild type,which may due to the lower transcriptional efficiency of separated promoter fragment of gene ST1 compared to the native promoter.In addition,development of stigma and style and the fertility were also recovered in all positive complementary plants.The results showed three constructions containing coiled-coil domain could complement the loss of function in stigma and style development in mutant st1,suggesting that coiled-coil domain may play a key role in the development of rice pistil.(2)Homozygous plants of mutant st1 were obtained by genotyping,and were used for screening candidate regulated genes of ST1 through RNA-seq.20 candidate genes related with floral development were selected and validated by Real-Time PCR.RNA-seq analysis results showed that 8 genes were up-regulated and 12 genes were down-regulated by gene ST1,and the expression levels of these candidate genes in mutant st1 were consistent with their reported functions during pistil development,e.g.ST1D1 and ST1D8 genes,suggesting that gene ST1 may directly or indirectly regulated these genes involving in rice pistil development.(3)RNAi vector of ST1 interacting protein ST1 P was constructed and transformed into rice variety ZH11 callus,and the progress is at the differentiation stage.In addition,positive and correct-edited transgenic plants for CRISPR editing of gene ST1 and XH5,another XHS gene which is up-regulated by gene ST1,were identified by positive check and sequence alignments.The works would provide important materials for further studying the function and molecular mechanism of gene ST1 in rice pistil development.