Biochemical Changes During Vernalization In Chinese Radish And Cloning Of LFY Gene Related To Florescence

Chinese radish(Raphanus sativus L.var. longgpinnatus Bailey) which comes from China is an important vegetable, the planted area is about 1,200,000 hektare, which locates the first three of that of all kinds of vegetables. At present, the key problem which restricts annual production and balanced supply of radish is the lack of bolting resistance germ plasm, this problem is also the essential reason of grievous expense with the matter of bolting that easily appears throuth producing. Therefore, prove up the mechanism of bolting resistance, combine the molecular breeding technology with the traditional breeding technology organically, and innovate bolting resistance germ plasm of radish, have an important theoretical and practical significance of speeding up the breeding process of radish. This paper studied the biochemical changes during vernalization in growing point of Chinese radish seedling, the foundation of efficient genetic transformational system of radish, and cloned the flowering key gene LFY. The main results were as follows:(1) during vernalization(2~4℃temperature treatment), the contents of soluble protein, free amino acid and soluble sugar in the growing point of two various cultivars changed in a similar tendency. The contents of soluble protein and free amino acid increased gradually, while the content of soluble sugar decreased. The contents of soluble protein and soluble sugar in the cultivar"Chang-Chun-Da-Xing"(late-bolting) were lower than in"Duan-Ye No.13"(early-bolting) obviously, while the content of free amino acid was on the other hand. The contents of gibberellin(GAs) and indole acetic acid(IAA) in the two cultivars had an obvious peak at the beginning of floral bud differentiation, and another peak can be abserved at the last stages of low temperature treatment.(2) Through studying on the factors of the effects on genetic transfonmation, on radish plant of"Duan-Ye No.13"the feasible vacuum infiltration genetic transformational system was screened out primarily: The vacuum inftltration time is 10min uninterrupted under 104pa negative pressure. Agrobacterium concentration is OD6000.8~1.2. Infiltration medium is 1/2MS inorganic composition, B5 organic composition, 6-BA 0.04mg·L-1 and AS 19.6 mg·L-1. In bud stage the buds are treated by the vacuum infiltration.(3) LFY gene fragment was amplified by RT-PCR after cloning and sequencing, the result revealed that it was highly homologous to Chinese cabbage LFY genes registered in Genbank(93%). Two LFY fragments in opposition were cloned into vector pUC19( molecular weight is 2686bp), and between them an intron of acetyl coenzyme A from wheat was inserted. Then the three fragments linked was inserted into plant expression vector pROK2 after chipped off by enzyme. Thereby the double strand RNA interference expression vector was constructed. The Chinese radish cultivar"Duan-Ye No.13"was transformed by vacuum infiltration genetic transformation which hoborting this ds-LFY RNAi vector.