The Identification Of Expansin Genes Role On Giant Embryo Formation Of In Rice

Giant embryo brown rice is rich in γ-aminobutyric acid(gamma-aminobutyric acid),protein,fat,amino acids,vitamins,minerals and other nutrients,so giant embryo rice is considered as a highly nutritious functional rice.The embryos of the giant embryo rice are 2 or 3 times larger than the embryos of other common embryos.In this laboratory,the giant embryo mutants were obtained by in vitro culture of " Chao 2-10" mature embryos.The whole genome expression profile of the caryopsis of "Shangshida 5" and "chao 2-10" were analyzed at 6 days,10 days and 14 days after flowering.We found that the expression of five expansin genes(OsEXPA1,OsEXPA4,OsEXPA8,OsEXPB2,OsEXPB18)were significantly up-regulated during embryonic development,the expansin gene may be a key gene involved in the cell size regulation process." Chao 2-10" rice and " Shangshida No.5 " giant embryo rice were used as materials to select the five expansin genes in rice,and role of expansin genes in the formation of giant embryo characters in rice were explored.The main contents and results are as follows:1.Chromosome mapping analysis of these 5 and other 13 expansin genes expressed in rice seeds,The results showed that the 18 expansin genes were unevenly distributed on the chromosome of rice.2.Ten CRISPR-Cas9 knockout vectors were constructed by two target genes designed by CRISPR-Cas,and two vectors were identified by Agrobacterium tumefaciens-mediated transformation."Shangshida No.5" and "Chao 2-10" rice.Among them,different numbers of OsEXPA4,OsEXPA8 and OsEXPB2 gene knockout positive plants were obtained,but the target gene was not mutated by sequencing.Transcribed level analysis and target gene coding sequence of three OsEXPB18 homozygous mutant plants and two OsEXPA1 heterozygous mutant plants were obtained.The results showed that the expression of target gene of OsEXPB18 homozygous mutant plant and OsEXPA1 hybrid knockout mutant was significantly down-regulated compared with wild type.The target gene was used to predict the protein sequence and functional domain prediction,We found that the two mutants of the target gene base insert or deletion of its corresponding expansion of protein important structural functional domains have different degrees of destruction;through field observation,we found that OsEXPB18 homozygous knockout mutant plants appeared fertility Low phenotype,So we have its pollen particles for iodine staining activity identification.respectively,The results showed that about 42%-70% of the pollen grains of OsEXPB18 homozygous knockout plants showed a Circular abortion phenotype compared to the control "Chao 2-10" rice,the seed count was observed and its seed setting rate was significantly lower than that of the control,and the volume of the whole seed was smaller.3.The overexpression of OsEXPA4 overexpressing transgenic plants was carried out.By comparing the changes of transcription level with the target gene,We found that the expression level of the target gene of overexpression of OsEXPA4 gene was significantly higher than that of wild type "chao 2-10" rice.Through the field observation,it was found that overexpression of OsEXPA4 overexpressed plants showed low dwarfing and low fertility phenotype,so we also have its pollen particles for iodine staining activity identification,The results showed that The fertility of OsEXPA4 overexpressing plants was significantly lower than that of wild type "chao 2-10" rice,The results showed that the pollen grains of OsEXPPA4 overexpressing plants decreased by about 56% compared with the wild type "Chao 2-10" rice,of which about 78% had lighter pollinated pollen phenotypes.By observing and analyzing the seeds,the majority of expression was found over OsEXPA4 sterile plants,and only an expression level of multiple genes upregulated lowest plant seed,and a seed rate(5%)was significantly lower than the control(90%),with the Compared with the control seed,the size of its whole seed did not change significantly,and the embryo volume became smaller.In this article,we demonstrate that the CRISPR-Cas9 system is effective in rice gene editing.It has a certain knockout efficiency for the target gene.At the same time we prove that the editing system which always played the role of an efficient editing in the progeny of genetic rice.At the same time,At the same time,we speculate that the expansins gene may be involved in the regulation of rice seed size,pollen development,seeding and other physiological processes,and different expansion factors may have different regulatory effects.This study has laid an important foundation for the further study on the molecular mechanism of seed embryo growth in giant embryo and the effect of the expansins gene on the pollen fertility of rice.