Proteomic Analyses During Bolting-related Leaf Color Change And Identification Of BrTUAs In Brassica Rapa

The Chinese cabbage(Brassica campestris L. ssp. pekinensis(Lour.) Olsson) is one of the most important vegetables in the genus Brassica of Cruciferae with the biggest cultivated area and longest cultivation history. The Chinese cabbage was called ‘Household Vegetable’ in northern China due to its high yield, low cost, easy cultivation and long storage. It plays a vital role in Shopping Basket Program.In recent years, off-season culture technology of Chinese cabbage developed rapidly and the cultivated area increased year by year. Because of the improper management and the climatic effect, premature bolting often happens which causes the lost of commercial and edible values of Chinese cabbage, reduce the yield, and caused huge losses to the farmer. Early bolting is a big problem for spring-cabbage cultivation and summer-cabbage production in high altitude. Meanwhile, unbolting or delay-bolting often occurs during seed production and adding-generation propagation which heavily reduces the yield and quality. Thus, revealing the molecular genetic mechanism of blossom especially bolting have profound meaning for breeding and cultivation.We had found that the Chinese cabbage have a leaf-color change period after the differentiation of flower bud. After the leaf-color changed from bright green to dark-green, flower bud elongated, then went into the bolting period. The leaf-color change seemed as a signal of bolting. The relationship between hormone, pigment, and leaf-color change was analyzed, meanwhile the gene expression level change of AP1、LEFY、FT was also investigated. This research aims to identify expression level of related gene by proteomic analysis, and reveal the underling mechanisms of developmental progress of Chinese cabbage on molecular level. The results are as follows:( 1)An inbred line of Chinese cabbage(C30) was analyzed by two-dimensional gel electrophoresis to figure out the proteomic change before and after the leaf-color change. 35 specifically expressed protein spots and 32 differential expressed protein spots were found. 17 specifically expressed proteins were selected for mass spectrometric analysis. Based on the Mascot scores, four valuable protein spots were identified: A. thaliana subunit E of vacuolar-type H+ transporter ATPase, the large subunit of Brassica rubisco, A. thaliana S-adenosylmethionine synthetase and canola tubulin α-2.(2)These clones were designated as Br VHA-E1, Brrbc L, Br TUA6 and Br SAMS, respectively and their expressions were analyzed by q RT-PCR using m RNA extracted before and after the leaf color change. The result indicated there were consistencies in protein and gene expression based on the protein analysis and q PCR results. It is suggested that these protein and gene may involved in the regulation of the bolting.(3)Using 6 TUA gene sequences of A. thaliana as query sequence to perform Blastp on Brassica Database, 12 Br TUAs were obtained. Through prediction of gene structure, function, and evolution of the 12 Br TUAs, it is found that the gene length, exon, intron, and promoter region changed along with the evolution of the genome. Among these 12 BrTUAs, only 5 gene code complete α micro-tubulin.(4)The result of phylogenetic analysis showed that Br TUA had closer relationship with AtTUA and Bn TUA, and the evolution of TUAs and the speices had the same pace.(5)Through analyzing promoter regions of the five genes, more than 60 cis-acting elements belong to 27 types were found. One photoperiod response element was found in Br TUA promoter region; different genes have great differences in low-temperature response element and hormone response element; one or more tissue specific expression elements were found in 5 BrTUAs’ promoter region, indicationg that different α-tubulins played different roles in regulation of growth and development of Chinese cabbage.(6)The resut of RT-PCR analysis showed that the five genes had differences in the temporal expression especially during the bolting-related leaf color change, indicating that Br TUA took part in the regulation of bolting and blossom in Chinese cabbage.(7)Spraying the the tubulin specific drugs Taxol as well as GA3, Methyl Jasmonate(MJ) to the vegetative growing point of Chinese cabbage during color-changed period, It is identified that GA3 and MJ could regulate the expression of Br TUA and bolting of Chinese cabbage.