| Chinese cabbage is a cross-pollination crop,which has obvious heterosis,and the application of hybrid seed production has become the primary measure for disease resistance,quality,and stable yield breeding of Chinese cabbage in the world.In the period research,a significantly different fragment in the male sterile line ‘A02' of Chinese cabbage was found by SSH technology.NCBI blastx analysied showed the fragment has the highest homology with Arabidopsis thaliana SKS13(At3g13400).So it is named as BrSKS13.Based on bioinformatics and expression pattern analysis,this study predicted the biological function of BrSKS13.The function of the gene was identified by the pollen phenotype and pollen germination of Arabidopsis homologous deletion mutant sks13,and the BrSKS13 gene was transferred into the homologous mutant sks13 to further verify its function.The main results are as follows:1.In order to determine the spatio-temporal expression pattern of BrSKS13,the expression of BrSKS13 in the roots,stems,leaves and buds of fertile and sterile Chinese cabbages was analyzed using RT-PCR technique.It was found that the expression of BrSKS13 was highly expressed in the buds of fertile plants;The expression of this gene in petal,Sepal,anthers and pistil of fertile flower buds showed higher expression in anthers;Then the expression of the gene in six stages of anthers was analyzed,and it was found that the expression level was up-regulated in a stage course of anther,It was shown that BrSKS13 is a flower bud development-related gene that may be involved in the development of anthers.qRT-PCR was used to analyze the fertile buds of sterile buds at 0h,1h,2h,4h,8h,12 h,16h,and 24 h after fertilization,and the results showed that BrSKS13 was still expressed and decreased along with the time going after pollination.When it reached 4h,the expression level increased rapidly and then decreased again.The result of in situ hybridization showed BrSKS13 was detected in the stigma after pollination and participated in the growth of pollen tubes.2.Mutant sks13 was purchased from the TAIR website and homozygous mutants were identified using the "Three primers".The phenotypes observation of homozygous mutants showed that there was no significant difference between the mutant plants and the wild type at the vegetative growth stage.When they entered the reproductive growth stage,the mutant plants were slightly smaller and the number of bolting was less than wild type.All the results indicated that the deletion of SKS13 might affect the reproductive development process of Arabidopsis.The pollen phenotype and pollen germination of homozygous mutants wereobserved,found that the absence of SKS13 could lead to the poor development of pollen and the abnormal germinate of pollen.The pot number and length was lower than wild type,indicating that the deletion of SKS13 affected the normal growth of Arabidopsis pods.With the over-expression vector,Brassica rapa BrSKS13 was transferred into homozygous mutants sks13 by Agrobacterium mediated transformation,and the transgenic plants were selected.The expression patterns analysis of the BrSKS13 gene in Arabidopsis revealed that the GUS signal appeared in anthers of transgenic plants,indicating that BrSKS13 is associated with anther-development.The transgenic plants,homozygous mutants and wild type were observed,the transgenic plants display the number of convulsions and scabs were increased,the length of pods were longer,the normal development of pollen than mutants,but there was no significant difference from the wild type.All these results showed that the insertion of BrSKS13 can restore the phenotype of the mutant sks13.In summary,BrSKS13 may participate in the process of pollen development and scarring. |
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