Overexpression Of Maize Cell Wall Invertase Incw2 Gene In Maize Kernels

High yield has been a major target in maize breeding programs, is a root way to solve the problem of food supply. Incw2 is a key regulator of the process of grain-filling, which is a key determinant of maize production. Incw2 encodes a cell-wall invertase INCW2, required for carbon partitioning during early grain-filling, regulates sucrose unloading, and then could increase yield potential through improved grain-filling. Incw2 shows a restricted expression pattern during the grain-filling in maize endosperm.In this study, rice glutelin Gil promoter and target gene Incw2 were cloned by homology cloning strategy, and constructed the plant expression vector 3300-Gtl-Incw2-bar driven with pGt1. Meanwhile, we constructed the plant expression vector 3300-27kD-Incw2-bar driven with p27kD, which was based on the expression vector 3300-27kD-nos-bar with zein gene p27kD (a endosperm-specific promoter of maize). And then both expression vector were introduced into maize embryonic calli and shoot apical meristems by Agrobacterum-mediated transformation, anticipating the success of overexpression of the cell wall invertase in maize endosperm, which can play the role of promoting the grain-filling, improving the grains yieid. The main results of this study are as fellow:1. Acquisition of a endosperm-specific promoter The Gtl promoter was isolated from rice genome DNA by homology cloning strategy. Sequencing results showed that the size of the promoter was 933bp, which has 100% homologous identical to the reported sequences of GenBank.2. Acquisition of target gene Regard genome DNA of maize inbred lines 18-599R as template, Incw2 was cloned. Sequencing results showed that the full-length of gene cloned was 2957bp, including 7 exons, coding 594 amino acids.-Results by Blast showed that the CDS of this gene had 99% homologous identical to the reported sequences of GenBank.3. Construction of expression vector The plant expression vetors 3300-Gtl-Incw-bar,3300-27kD-Incw2-bar were repectively constructed, in which Incw2 was driven by endosperm-specific promoter pGt1 and p27kD. Results of sequence analysis showed that the target gene had no mutations during the process of construction of expression vectors.4. Establishment of transformation system The two expression vectors with target gene Incw2 were introduced into calli transformation system of the maize inbred line 18-599R by Agrobacterium-mediated transformation, and established successfully shoot apical meristems transformation system of inbred lines 18-588W and R08 by Agrobacterum-mediated approach.5. Acquisition of resistance transgenic plants Transgenic resistance plants were obtained after screening with herbicide Basta.38 and 13 resistant plants of To generation were obtained from the resistant calli transformed with 3300-Gt1-Incw2-bar,3300-27kD-Incw2-bar respectively; 147 and 58 resistant plants of To generation were obtained from the shoot apical meristems transformed with3300-Gt1-Incw2-bar,3300-27kD-Incw2-bar respectively.6. Acquisition of positive transgenic plants 34 and 13 To resistance plants transformed with 3300-Gt1-Incw2-bar,3300-27kD-Incw2-bar respectively were certified to be positive by PCR detection using gene-specific primers, which were designed combined with the upstream promoter and downstream target gene sequences. The PCR results of the T1 transgenic plant lines indicated that exogenous gene exhibited stable inheritance, did not occer the phenomenon of gene missing.7. Valid expression of target gene 5 T1 transgenic plant lines kernels transformed with 3300-Gt1-Incw2-bar selected randomly were tested by semi-quantitative RT-PCR. The result indicated the expression quantitys of Incw2 of 4 lines were higher significantly in comparison to the control, and there were differences between plants. Cell wall invertase activity in the above plants was the same with the result of semi-quantitative RT-PCR. Compared with the control, Cell wall invertase activity of the 4 plants above exhibited distinct increase, and the top increased 69.08%, which indicated that the foreign gene Incw2 has been expressed validly.8. The yield-related traits of the T1 transgenic plant lines were improved The yield-related traits of the T1 transgenic plant lines were investigated at 12 days after pollination. The result shew that the photosynthetic efficiency, chlorophyll content, leaf nitrogen concentrations were all much higher than the control. Meanwhile, the green leaf number was also much more than the control of varying degrees during harvest time, but the difference of the plant height and ear height were not obvious compared to the control.100-grain weight were higher than the control at different level. The results show that the Incw2 gene could increase yield potential through improved grain-filling.