Overexpression Of Glutamine Synthetase (Gln1-3) Gene In Maize Inbred Line 18-599(R)

As one of the three most important crops in China, maize plays an important role in food supply, economic income and feed crop supply. With the quantity demand of maize rising dramatically, the breeding of high yield, high quality and efficient maize variety is the focus of work.Plants have the most quantity demand of nitrogen in essential mineral elements. Uptake and assimilation of nitrogen is the most important physiological process, so combining traditional breeding strategies of molecμlar breeding with transgenic technology to improve the assimilation of inorganic nitrogen and organic nitrogen such as Glutamine and glutamic acid efficiency and enhance nitrogen absorption and utilization of plant capacity,is to cultivate high-yielding varieties of high N uptake and use efficiency is one important way to a series of questions. Glutamine synthetase is the main rate-limiting enzyme which is used as plant nitrogen assimilation. Numerous studies reveal that glutamine synthetase can enhance plant absorption and utilization of nitrogen to improve the photosynthetic efficiency and increase production, what's more, maize Gln1-3 can promote the transfer of nitrogen in the whole plants to kernel and then been assimilated to increase the number of kernels, therefore, overexpression of glutamine synthetase gene Gln1-3 in maize is likely to improve kernel production.In this study, ORF of Gln1-3 is seperated by homology cloning from the maize inbred line 18-599 (red), two recombinant vector are constructed with constitutive promoter CaMV 35S and Ubiquitin, named pcambia1301-Gln1-3and Ubiquitinl301-Gln1-3 respectively followed by Agrobacterium-mediated transformation into maize embryogenic calli of inbred line 18-599 (red).Resistant plants are obtained through antibiotic filter and regeneration differentiation. Several methods, such as PCR, RT-PCR and glutamine synthelase activity determination are used, which demonstrates that Glnl-3 has been integrated into maize genome. Trait analyse of T1 generation indicates that transgenic plants tend to increase producion. The main resμlts are as follows:1. ORF of Gln1-3 is obtained by homology cloning from maize inbred line 18-599(red) with full size of 1051 bp, the homology of nucleotide shows that it is 99% genetically identical to mRNA of maize gs1-3(X65928.1) and there are two base mutations, however, the homology of amino acid shows there is no difference compared to the original sequence, so the two base mutations have no effect on the active site of the enzyme.2.We have successfμlly constructed plant expression vector Ubiquitin1301-Gln1-3 and pcambial301-Gln1-3, respectively, with the Ubiquitin promoter which has strong expression in monocots and the CAMV 35S promoter which has strong expression in dicots. There is no mutation and with right inserting directon through restriction endonuclease reaction and sequencing.3.20,000 embryogenic calli of inbred line 18-599 (red) were transformed, and 6 resistant plants were obtained by hygromycin selection, among which, all of them are transformed with Ubiquitin1301-Gln1-3.4. TO transgenic seedlings were PCR tested which showed that the 6 resistant plants were positive and revealed that the exogenous gene had been integrated into the genome of maize. In addition, glutamine synthelase activity determination of TO leaves showed that it had been improved differently, however, only 4 trangenic plants' seeds were obtained because of the bad weather.5. PCR test of 4 T1 lines shows us that 4 transgenic plants from 2 of 4 lines are positive, we find the expression level of transgenic plants is higher than the control using RT-PCR test in step with the resμlt of glutamine synthelase activity determination.6. Photosynthetic efficiency and nitrogen content test of the T1 transgenic plants including after pollination showed an higher value compared to WT. We can draw a conclusion that overexpression of Glnl-3 can increase efficiency to transfer nitrogen of other parts of plants to kernel.7. Yield-related traits measurement of 4 transgenic plants showed us ear diameter and hundred grain weight of 2 had been improved significantly, so we can say Glnl-3 coμld lift maize yield.