Study On The Molecular Mechanism Of Pistil Development Of The Super Silique Mutant In Brassica Napus L.

Oilseed rape(Brassica napus L.)is an important oil and economic crop in China.For oilseed rape and many other crops,the development of flower organ is an important event related to their yield and economic value,in which the female reproductive organ,pistil,can eventually develop into fruit,having a direct and decisive impact on their seed yield and quality.The research on the mechanism of pistil development mainly depends on Arabidopsis mutants.However,up to now,these mutants are all defective or female sterile mutants.In the previous work of this study,a mutant Bnspt(Brassica napus super pistil mutant)with super long silique phenotype was obtained.In this study,this mutant was used as research material to study the development of pistil.The main results are as follows:(1)Firstly,the phenotype of Bnspt mutant was investigated.The results showed that,compared with untransformed plants(CK,Control Check),the Bnspt mutant with super long silique phenotype showed an increase in seed volume,with a 1000 seeds weight of 6.61 g,which was more than twice that of CK.However,there was no significant change in the seed number of per silique,silique number of per branch,the number of primary branches and plant height.(2)Furthermore,RNA-Seq method was used to analyze the differentially expressed genes(DEGs)between Bnspt and CK.According to the criteria of FDR<0.05 and |Fold Change|≥2,9022 genes were found to have significant changes in the expression level,including 4795 up-regulated genes and 4227 down-regulated genes.Go enrichment results showed that the largest number of DEGs enrichment was in the category of “cellular component”,including 7,592 genes.The analysis of KEGG pathway enrichment showed that the DEGs were mainly concentrated in the “Protein processing in endoplasmic reticulum”,Linoleic acid metabolism” and “Pentose and glucuronate interconversions”,in which the pathway with the most abundant DEGs was“Protein processing in endoplasmic reticulum”.(3)We further analyzed the DEGs related to hormone signaling pathway.The results showed that there were differentially expressed genes in the signal pathways of auxin,cytokinin,gibberellins and brassinolides,suggesting that these hormones play roles in the pistil development of Bnspt.(4)FPNI-PCR(Fusion primer and nested integrated PCR)was used to amplify the flanking sequence of T-DNA insertion of Bnspt mutant genome,and the inserted gene was cloned and named Bn SPT.Sequence analysis showed that Bn SPT encodes a protein composed of 302 amino acids.Tissue pattern analysis showed that the expression level of Bn SPT was extremely significant in fruit and flower,and was,respectively,71.92 times and 11.01 times of that in stem.These results suggested a role for Bn SPT in the development of fruit and flower.In addition,we further found that the expression of Bn SPT in the pistil of the Bnspt mutant was significantly higher than that of CK.(5)The subcellular localization analysis of onion epidermis transient expression experiment in mediated by Agrobacterium showed that Bn SPT was located in the nucleus of onion epidermis cells,indicating that Bn SPT played a role in the nucleus.(6)In view of the fact that the expression of the mutant Bnspt was significantly higher than that of the CK,we further created transgenic oilseed rape plants overexpressing Bn SPT for phenotype observation.We constructed the overexpression vector of Bn SPT,and obtained three transgenic oilseed rape lines overexpressing Bn SPT by Agrobacterium mediated transformation of oilseed rape hypocotyls.In the future,we will carry out further phenotypic observation and related research on the positive transformants of these three lines.