Cloning Of HN-ZMZ-1 Gene And Research On Transformation Of Agrobacterium Tumefaciens

Soybean is one of the main food crops in China and the main source of protein and vegetable oil for daily life.Using genetic engineering technology to breed soybean varieties with high yield and high quality can alleviate the dependence on soybean import in China to a certain extent,which is of great significance to meet the increasing yield and quality requirements of people.Photosynthesis was the key factor for yield formation.Sedoheptulose-1,7-bisphosphatase（SBPase）was the key enzyme controlling carbon flow in the Calvin cycle of photosynthesis,and was also related to the regeneration of Ru BP and the synthesis of carbohydrates.In this study,soybean varieties Shennong 9 and Williams 82 were used as test materials to optimize the agrobacterium-mediated soybean genetic transformation system to improve the induction rate of resistant cluster shoots.Then,the optimized agrobacterium transformation system was used to transform the cloned soybean SBPase gene,and the gene was named HN-ZMZ-1.Finally,the positive T0 generation plants were preliminarily verified.This study will provide the basis for the establishment of efficient and stable soybean transformation system,the in-depth study of HN-ZMZ-1 gene function,and the practice of high-yield soybean breeding.The main results are as follows:1.The effects of soybean cotyledon node scratch and non-scratch treatments on the induction of resistant cluster shoots were studied.In the process of agrobacterium infection on explants,the induction rate of resistant cluster shoots of soybean could be improved by 0.5mp vacuum osmosis for 3 min.In the co-culture process,dark culture for 3 days and light culture for 2days were beneficial to the induction of resistant cluster shoots.When the concentration of6-BA was 1.75 mg′L^-1,the induction rate of resistant cluster shoots reached the highest.2.In this study,SBPase gene was cloned from the leaf of soybean genotype Williams 82 and named HN-ZMZ-1.Sequence analysis showed that the gene was 1,399 bp in length and had a coding frame of 1,164 bp,encoding 387 amino acids in total.The molecular formula was C₁₈₆₄H₂₉₅₁N₄₈₉O₅₇₃S₁₁.The protein molecular weight was 41.7k Da and the isoelectric point was 6.05.Meanwhile,the plant expression vector pearleygate100-HN-ZMZ-1 was constructed,which contained the screening marker BAR gene and Kan,and was transferred into agrobacterium tumefaciens strain GV3101 for subsequent genetic transformation of soybean.3.The agrobacterium strain GV3101 containing the expression vector pearleygate100-HN-ZMZ-1 was transformed into Williams 82,and 52 resistant plants were obtained after induction of resistant cluster shoots,elongation,rooting,hardening and transplanting.A total of 6 T0-generation positive plants were obtained by PCR,including 4 HN-ZMZ-1 gene transfected（Tr）and 2 empty vector transfected（Tr-CK）.Fluorescence quantitative PCR analysis of HN-ZMZ-1 transgenic plants showed that the transcriptional expression of HN-ZMZ-1 gene in transgenic plants was significantly higher than that of the control,which was 1.58 times,3.29 times,3.9 times and 3.22 times,respectively.