Expression Analysis And Cloning Of ACC Synthase Gene In Watermelon (Citrullus Lanatus)

Plant sex differentiation is a highly complex physiological and biochemical morphogenesis process which under the control of a certain genetic background. It is a good experimental system to study on molecular biology of plant development and regulation of gene expression, it is also one of the major issues to be explored. Because of the sex differentiation specificity, the flowers of Cucurbitas are diversity, and the sex differentiation of melon crops is vulnerable to be impacted by external factors. So it became a classic experiment material on sex determination research. As an important Cucurbita crop, the sex differentiation of watermelon was affected significantly by application of ethylene, so our research focus on the relationship between ethylene biosynthesis, signal transduction and sex differentiation.The practical significance of the research is obvious which can detect the molecular mechanism of sex differentiation in plants.According to the sequences of watermelon ACC synthase gene(ACS) in Genebank we designed primers, and then ACS genes/fragments of subgynoecious of watermelon had been cloned. In order to obtain the more information about the relationship between ACS gene and sex differentiation of watermelon, the present experiment was carried out to study the expression of ACS gene both different organs and different developmental stages of flower by semi-quantitative RT-PCR and real-time quantitative PCR.The results obtained as following:(1) Ethylene inhibited female flower differentiation in watermelon when seedling treated by 50mg/L and 100 mg/L ethephon, higher concentration ethephon(300mg/L, 500mg/L) produced phytotoxicity on watermelon.(2) Four ACC synthase gene/fragments, Cit-ACS1, Cit-ACS₂, Cit-ACS₃ and Cit-ACS4, had been cloned using the total RNA extracted from the stem apex of subgynoecious watermelon. Sequence homology between ACS genes in subgynoecious line and that as reported in normal watermelon was 99.8-100%. Phylogenetic tree of ACS gene was constructed, and the secondary and tertiary structures of protein encoded by Cit-ACS1 gene were also analyzed in this experiment. (3) The results of expression of Cit-ACS1-4 genes in watermelon showed that: the expression levels of Cit-ACS1 gene in flower organs except the male flower of normal sex type line TD-2 were higher than that in subgynoecious line TD-1. Moreover, the expression of Cit-ACS1 gene was upregulated after treated with 50mg/L ethephon for two weeks, while female flower ratio was decreased. We concluded that the lower level expression of Cit-ACS1 gene will be beneficial to female flower differentiation in watermelon, whereas the higher level expression of Cit-ACS1 will inhibite female flower differentiation. Cit-ACS2 gene only expressed in the vegetative organs (young leaves and tendrils) in watermelon TD-1 and TD-2, sex differentiation was less affected by Cit-ACS2. The expression level of Cit-ACS3 gene in 2-4mm, 4-6mm female flower buds of TD-1 were 42.9%, 19.4% of TD-2, respctively, and low-level expression of this gene was conducive to female flower generation. The expression level of Cit-ACS4 gene in flower bud and 2-4mm male flower of TD-1 was lower than that of TD-2, so high-level expression of Cit-ACS4 gene may be beneficial to the male flower differentiation. The expression abundance of Cit-ACS1 gene was higher than other ACS genes, and it played a major role in the process of sex differentiation in watermelon.(4) The signal transduction pathway from Cit-ACS genes to sexual differentiation of watermelon was predicted.