Analysis Of BcERF-B3 Gene In Stamen Mutation And Its Maintainer Line Of Non-Heading Chinese Cabbage

Non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino),which is originated from Chinese Mainland,is a favorite vegetables for Chinese.In recent years,the demand for production and quality of non-heading Chinese cabbage is increasingly required.However,due to few stable male sterile varieties,it is hard to take full advantage of heterosis for excellent germplasm,so accessing to male sterile line with fully sterility has become one of the most urgent task now.Stamen transformed into petal-like structures,which causes entirely pollen-less,is used for cross breeding among non-heading Chinese cabbage.In this study,the stable stamen-petalody line 'subai 2' with its maintainer line were utilized as research material.Firstly,the metabolic physiological indexs of two kinds of material were measured,then an ethylene-responsive factor gene,which had been got from mutant by cDNA-AFLP technique,was cloned,named BcERF-B3,from non-heading Chinese cabbage by RT-PCR,and the exprsssion level of this gene in different tissues and development stages were explore with qRT-PCR,a recombinant overexpression vector was constructed and transgenic verified to identify,the function of the gene.In addition,we had conducted the prokaryotic expression,for further exploring the role of this gene and it's downstream genes.The results were shown as below:1.Studies on morphological and biochemical traits of mutant and its maintainer line of non-heading Chinese cabbageBased on stamen petalody mutant line with genetic stability and its maintainer in non-heading Chinese cabbage,we detected the morphological differences and the protective enzymes activity,content of MDA,soluble sugar and soluble protein during different flower development stages of two cultivars.Morphological results showed that the most obvious difference of floral organs were the stamens of mutant with complete deletion,instead of a corresponding increase of petals in the same location,we concluded that this stamen mutant was floral homeotic mutant line;physiological characteristics analysis indicated that the activity of MDA and three enzymes in mutnat were significant higher than maintainer at middle and later stages of flower development;during different flower development stages,the content of soluble sugar and protein in mutant was lower than the contents in maintainer line,but significantly higher at middle stages of flower development.2.Clone and expression of BcERF-B3 in non-heading Chinese cabbageBased on the sequences of differential fragment,we cloned an ERF gene,named BcERF-B3,from non-heading Chinese cabbage by RT-PCR.Then the nucleotide and amino acid sequence of BcERF-B3 was analyzed by related bioinformatics software.Sequence analysis showed that the open reading frame sequence of BcERF-B3 gene was 807 bp,encoded 268 amino acids residues;RT-PCR results showed that BcERF-B3 gene have a highly expression in stamen,petal and carpel in mutant than maintainer lines,transcript abundance in stamen of mutant changed most significantly in comparison to maintainer,which suggested this gene might play important roles in stamen morphological change in non-heading Chinese cabbage;quantitative RT-PCR indicated that BcERF-B3 gene had higher expression level in middle stages of flower development(0.5 mm<bud diameter<2 mm)of mutant,and external ethylene increased the gene expression,while external AgNO3 inhibited its expression.These results suggested that the gene might involve in stamen mutant.3.Construction of overexpression vector of BcERF-B3 and its expression level at different stressss of two linesTo know the expression profile of gene in different abiotic stresses,qPCR was uesd to detect the transcript level of BcERF-B3 mRNA.After abscisic acid,methyl jasmonate and cold treatment,the expression levels of BcERF-B3 gene in mutant could be up-regulated quickly.These work indicated BcERF-B3 might be responsive to stress,then plays an important role in the regulation of flower mutant in non-heading Chinese cabbage;an efficiently overexpressing vector of pEarleygate103-BcERF-B3 was constructed and transformed into agrobacterium with electric shocking.Our study helps understanding the function of BcERF-B3 gene and exploring the mechanism of BcERF-B3 protein regulation in non-heading Chinese cabbage;furthermore,this study provides a foundation for find optium sterility line by molecular breeding.4.Expression of non-heading Chinese cabbage BcERF-B3 Gene in prokaryotic cellThe full-length of BcERF-B3 ORF,we got,was introduced directionally into prokaryotic expression vector pET30a by enzyme digestion,then the recombinant expression vector pET30a-BcERF-B3 was transformed into Escherichia coli host strain BL21(DE3)for further expression induced by Isopropylthio-?-D-galactoside(IPTG).The IPTG concentration and temperature of expression were optimized using SDS-PAGE.An efficiently prokaryotic vector of pET30a-BcERF-B3 was constructed and transformed into BL21(DE3)with hot shocking;the SDS-PAGE results indicated that fusion protein was expressed with molecular weight of 35.4 KD,but the expression level was low.The result is consistent with the 29.6 KD protein which was encoded by BcERF-B3 gene.The optimal expression condition was set as 1 mmol/L IPTG,under 37? induced temperature,then was lysed by sonication.The prokaryotic expression system establishment lay the foundation for further investigating the function of the BcERF-B3 protein in non-heading Chinese cabage.