| Chinese cabbage（Brassica rapa L.ssp.pekinensis）is a vegetable crop of the genus brassica in the cruciferous family,which is characterized by heterogamous pollination of hermaphroditic flowers and significant hybrid advantage.Using male sterile lines to prepare Chinese cabbage hybrids,the purity of hybrid can achieve 100%,which is more conducive to the protection of the rights of new varieties,and is an ideal way to exploit hybrid advantage.In order to further explore and identify male sterility genes,this study firstly constructed a functional analysis system for Chinese cabbage using two gene families CBL and CIPK,as examples,while using 3A sterile lines as sterile sources,and using transcriptome sequencing technology to analyze the expression of pure sterile lines（18WH-2A）and pure fertile lines（18WH-2B）at the RNA level in four different periods of flower buds.In addition,the BSA data were combined to screen fertility candidate genes,and CRISPR/Cas9 technology was used to knock out the related genes and resolve the functions of the fertility-related genes.This will provide a theoretical basis for unraveling the abortive mechanism of this superior male sterile resource,and will be of great significance for the artificial creation of superior male sterile lines.The main findings of the study are as following:1.18 BraCBL genes and 47 BraCIPK genes were identified using Arabidopsis CBL and CIPK sequences,and the construction of their respective phylogenetic trees revealed that BraCBL could be divided into three subfamilies and BraCIPK into two subfamilies.Analysis of the NAF motifs of the CBL and CIPK gene families revealed that all BraCIPK members,except BraCIPK4.1,have NAF/FISL structural domains and that there are differences in the NAF sequences between the duplicated genes.2.Analysis of the expression profiles of BraCBL and BraCIPK genes under different stress conditions showed different expression trends between the different duplicated genes during treatment with Mg2+,K+and low temperature treatments.The interaction profiles in yeast two-hybrid assays,suggesting functional divergence of the duplicated genes during the long-term evolution of Chinese cabbage,were further validated using BraCIPK23.1,BraCIPK23.2 and BraCIPK23.3 transgenic Arabidopsis thaliana for potassium deficiency treatment.3.The latest A03 reference genome was used to analyze the BSA data of Chinese cabbage,and the ED algorithm was used to perform association analysis of the regions.A total of four candidate differential trait regions were screened,located at both ends of chromosome 4 and in chromosome regions 7 and 9,with a total length of 9.76 Mb,containing a total of 1577 genes,including 440 non-synonymous mutations and 104shift mutations in the mixed pools.4.The transcriptomes of four anthers from sterile and fertile lines of Chinese cabbage at different stages of development were sequenced and subjected to differential gene analysis.The results were showed as follows:A1 vs.B1,with 138 genes upregulated and 289 genes downregulated;A2 vs.B2,with 1048 genes upregulated and531 genes downregulated;A3 vs.B3,with 2381 genes upregulated and 2227 genes downregulated;and A4 vs.B4,with 1673 genes upregulated and 3769 genes downregulated.5.The thickness of the tapetum was counted for each period,and the differential genes in eight samples（TPM values>1）were used to perform the WGCNA analysis.The genes were divided into ten modules according to the similarity of their expression patterns to analyse the correlation between genes and traits.The highest correlation coefficients between genes and traits were observed in the modules Turquoise,Midnightblue and Purple（-0.76,P-value=0.00002;-0.61,P-value=0.002）,with the Turquoise module containing 2630 genes,the Midnightblue module containing 52genes,and the Purple module containing 1025 genes.6.The BSA and WGCNA results were intersected to obtain 43 candidate genes,and BAA09g40540,BAA09g41000,BAA07g09190,BAA09g42010 were selected for CRISPR/Cas9 gene editing in Arabidopsis.The T1 generation plants were sequenced and the gene editing was successful,but subsequent screening of pure plants and observation of phenotypes are still needed.The results of the q RT-PCR validation test for the above genes were consistent with the transcriptome data,confirming the reliability of the transcriptome data.In summary,this study has mined the transcriptome and CRISPR/Cas9 to identify candidate genes controlling fertility in the near-isogenic line 18WH-2 material of Chinese cabbage,providing a theoretical basis for the regulation mechanism of male infertility and a molecular basis for the subsequent transformation of such male-sterile materials. |
