| In China, the main foreign insect-resistant genes of transgenic cotton is Bt gene, it has good resistance on Lepidoptera pests such as bollworm and pink bollworm. However, with transgenic cotton spreading through large areas and many years of field application, the scientists found that the decreased uses of pesticides in recent years led to aphids, Lygus stink bugs, cotton whitefly and other non-major pests increased sharply. In addition, Cotton Verticillium wilt, the most serious disease, has dramatically reduced the cotton productions. Pathogenesis of Verticillium wilt is complex, so the progress is slow with low-efficient breeding and lack of highly resistant varieties currently. Cotton pigment glands have gossypol and other secondary metabolites deposition, which has good resistance to a variety of pests and diseases. Therefore, we want to improve the content of gossypol by effectively genetic engineering regulation for secondary metabolism, which would have important theoretical and practical value.Cadinene synthase catalyzes farnesyl pyrophosphate (FPP) cyclization to form (+)-δ-cadinene, which is the rate-limiting step in the pathway of gossypol synthesis. Cadinene is a common precursor of gossypol and all its derivatives. In this study, we cloned Cdn gene from the upland Y18R and analyzed its cDNA and genome sequences. The genome sequences of Cdn gene are 2700bp, including six introns, and its sheared transcript are 1665 bp, encoding 554 amino acid residues. This gene encodes a protein with a terpene synthase (cyclase) active conservative region - DDXXD. Analysis of the protein phylogenetic tree show that the gene belong to cadinene synthase C subfamily. The Overlap PCR method was applied to making two HindⅢrestriction sites of Cdn gene passivated, and the process did not change the encoded amino acid sequences.We connected the Cdn gene to plant expression vector pBI121, with the constitutive promoter CaMV35S and green tissue-specific promoter Psbp driving its expressions, and successfully constructed two plant expression vector pGBI-CaMV35S-Cdn and pGBI-Psbp-Cdn. In addition, we also constructed a RNAi interference vector pK7GWIWG2(I),0-571 by Gateway. We transformed the three constructed vector by Agrobacterium mediated to cotton hypocotyls and conducted tissue culture. We got nine 35S transgenic (pGBI-CaMV35S-Cdn) callus lines and seedlings, two P transgenic (pGBI-Psbp-Cdn) callus lines and seedlings, four R transgenic (pK7GWIWG2 (I), 0-571) callus lines.Ream-Time PCR analysis showed that mRNA expression of Cdn gene in callus lines 35S-1 to 35S-7 are 8.97-9.64 times as non-transgenic control,s; mRNA expression in 35S-8 and 35S-9 lines are 2.88 and 3.34 times as non-transgenic control,s, respectively. The mRNA expression of Cdn gene in callus lines P1 and P2 are 1.33 and 1.28 times as control,s. Transforme by plant expression vector showed that expression levels of Cdn gene transcription have increased. The mRNA expression of Cdn gene in callus lines R-1 to R-4 are 0.43,0.49,0.55 and 0.41 times as the control,s. The results suggest that Cdn gene transcription was significantly inhibited after RNA interference.Measuring gossypol content of callus, we found 35S-1 to 35S-7 lines are 61% -86% higher than that in non-transgenic control. Gossypol content of 35S-8 and 35S-9 lines are 41% and 49% higher than that in non-transgenic control. P1 and P2 lines are 32% and 18% higher than control. Gossypol content in callus R lines are 96%, 108%, 109% and 92% of non-transgenic control. It can be seen that gossypol content of 35S and P transgenic lines were higher than non-transgenic control, and R lines were very close to the control, not Reducing as expected. With the previous result for mRNA expression level of Cdn gene in callus lines, we found that increased mRNA expression level of Cdn gene has a definite positive correlation with the gossypol content to some degree. In other words, the higher Cdn gene express, the little more gossypol content will be. Though mRNA expression level of Cdn gene in R lines was reduced by 50%, gossypol content was consistent with the control, infering that may be due to redundant complementary by other Cdn subfamily.The synthesis of gossypol is a cumulative process, the gossypol content is generally higher in seedling stage than in callus stage. In the seedling stage, gossypol content in 35S-1 to 35S-7 were 17% -38% higher than the control, 35S-8 and 35S-9 were 16% and 7% higher. P1 and P2 were 12% and 17% higher.Inducing callus into seedlings, gossypol content in 35S and P lines, though still higher than the control, but the gap is reduced. Although the gap between P lines and control reduced, the decrease is lower than the 35S lines. Preliminary judging in the green seedling tissue-specific promoter Psbp start to work gradually.Gossypol content in browning callus were slightly higher than the corresponding normal ones. The three transgenic lines of callus is no great difference between them. The rate of control were 97%, indicating that Agrobacterium infection process may reduce the rate of cotton tissue culture callus. At this time, gossypol has little influence on the callus tissue. In the seedling stage, there would be some vitrifications. Gossypol content in vitrifications were detected 5% higher than normal seedlings. Whether gossypol content has a negative impact on glass formation will be tested by further experiments. The normal germination rates of 35S and P transgenic callus lines were about 10%, much lower than 53% of non-transgenic control. We speculate that gossypol has little effect on the callus, but in the process of tissue culture, gossypol accumulation ultimately inhibited the emergence of callus.In summary, this research provide theoretical basis for the ideal to cultivating a new spieces with highgossypol plant&low-gossypol seed. |
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