Glucosinolates-regulated Factors And Cloning Of CYP79F1 Gene Related To Aliphatic Glucosinolate Synthesis

Glucosinolates are one group of plant secondary metabolites mainly found in Cruciferae. Their enzymatical degradation products are responsible for the characteristic flavor, pathogen defense system and insect attractants of Cruciferae. And glucosinolates have been identified as chemopreventive agents for human beings with in-depth study. Therefore, how to get more beneficial glucosinolates from Cruciferae has become a hot research topic in recent years. Pakchoi(Brassica campestris L. ssp. chinensis var. communis) is one of the common and extensively cultivated leafy vegetable in China. Two pakchoi cultivars, Shanghaiqing & Youdonger, are selected as materials to study the effects of sulfur, sugar, salt and some plant growth regulators on the profiles and contents of glucosinolates. The gene CYP79F1, which play an important role in the synthesis of aliphatic glucosinolates, has been cloned from pakchoi by RT-PCR. This may help us to alter the aliphatic glucosinolate profiles by genetic methods. The main results as follows:1. Two cultivars of pakchoi (cv. Shanghaiqing and Youdonger) hydroponically in nutrient solution supplied with two levels of sulfur (0.0558 mM as sulfur deficiency and 1.0058 mM as sulfur sufficiency, respectively). Insufficient sulfur supply significantly inhibited the plant growth. What's more, sulfur deficiency reduced the total glucosinolate content and most of the individual glucosinolate content in both cultivars. However, two cultivars responded to sulfur deficiency differently in glucosinolate metabolism. Shanghaiqing was significantly affected by sulfur deficiency with a pronounced decrease in total glucosinolates and aliphatic glucosinolates. In contrast Youdonger exhibited a larger decrease in indole and aromatic glucosinolate content than Shanghaiqing.2. Effects of sucrose and glucose spray on plant growth and glucosinolates content were investigated in Shanghaiqing. The experiment was treated as the spray of pure water (control),0.1 M sucrose,0.2 M sucrose,0.1 M glucose and 0.2 M glucose. The results showed that the spray of sucrose and glucose did not significantly affect biomass of pakchoi, as compared to the control. The contents of total aliphatic glucosinolates, total indole glucosinolates and total glucosinolates were significantly increased by 0.1 and 0.2 M sucrose treatments.0.2 M glucose treatment increased the contents of total aliphatic glucosinolates, total indole glucosinolates and total glucosinolates. However, all individual glucosinolates content were not significantly influenced by 0.1 M glucose treatment. aromatic glucosinolate content was not significantly changed by sucrose and glucose treatments.3. Investigate the contents and composition of glucosinolates in Shanghaiqing exposed to 0,50 and 100 mM sodium chloride (NaCl). The results showed that salt stress significantly decreased both fresh and dry weight of pakchoi shoots and roots, as compared to the control. Under 50 mM NaCl the contents of total glucosinolates, aliphatic and indole glucosinolates in pakchoi shoots were significantly increased. Under 100 mM NaCl the content of indole glucosinolates was significantly increased and aromatic glucosinolate content was significantly decreased. However, NaCl treatments reduced most individual and total glucosinolate contents in pakchoi roots.4. The effects of different concentrations (0,10,50,100 and 200μl.l-1) of ethephon on glucosinolates contents were studied in Shanghaiqing and Youdonger. The results showed that spray of ethephon reduced biomass of pakchoi. The content of total aliphatic glucosinolate was significantly increased at 10μl.l-1 treatment in Shanghaiqing; that was at 100μl.l-1 ethephon treatment in Youdonger. The total indole glucosinolate content was not significant influenced under different concentrations of ethephon treatments in Youdonger. However,100 and 200μl.l-1 ethephon treatments significantly increased the content of the total indole glucosinolates in Shanghaiqing. However, two cultivars responded to ethephon differently in aromatic metabolism.5. The effects of different concentrations (0,5,10 and 20 mg.L-1) of abscisic acid (ABA) on glucosinolates contents were studied in Shanghaiqing. The results showed that spray of ABA did not significantly affect biomass of pakchoi, as compared to the control. Exogenous ABA application exhibited concentration dependent manner on individual glucosinolates and total glucosinolates. Total aliphatic glucosinolates and total glucosinolates decreased with increasing concentration of exogenous ABA. However,10 mg.L-1 ABA treatment significantly increased the content in pakchoi shoots. The content of aromatic glucosinolate significantly increased at 20 mg.L-1 ABA treatment.6. The effects of different concentrations (0,0.2,5 and 20 mg.L-1) of cytokinin (CTK) on glucosinolates contents were studied in Shanghaiqing. The results showed that spray of CTK increased the biomass of pakchoi with increasing concentration of exogenous CTK. The content of total aliphatic and total glucosinolates reached maximum at 20 mg.L-1 CTK treatment. However,0.2 mg.L-1 CTK treatment increased the content of the total indole and aromatic glucosinolate.7. The effects of different concentrations of NAA, sprayed by one or two times, and IAA on glucosinolates contents were studied in pakchoi shoots. The results showed that spray of NAA and IAA increased the biomass of pakchoi with increasing concentration of exogenous NAA and IAA. The content of aliphatic glucosinolates, aromatic glucosinolate and total glucosinolates content reached maximum at 20 mg.L-1 NAA by one time spay treatment. Most individual indole glucosinolates content reached maximum at 50 mg.L-1 NAA treatment. Most of individual glucosinolates and total glucosinolates reached the maximum level at 10 mg.L-1 NAA by two times of spray treatment.5-20 mg.L-1 IAA treatments significantly increased the content of most individual aliphatic and total glucosinolates, however, most individual indole glucosinolates reached maximum at 5 mg.L"1 NAA treatment. In addition, aromatic glucosinolate content was increased under IAA treatments.8. According to the sequence of Arabidopsis thaliana, Chinese cabbage and cabbage, we designed specific primers, and cloned the cDNA of CYP79F1 from Youdonger. The coding region had 1623bp, and the deduced protein had 540 amino acids and its molecular mass was 61.468kDa. In Genebank only three homologous genes which are from Chinese cabbage, cabbage and Arabidopsis have been found, the similarity was 99.07%,97.57% and 83.36%, respectively, compared to pakchoi.