| Salinity and drought have became the cosmopolitan problem, which threaten seriously the development of agriculture and animal husbandry, and even the security of whole ecosystem. Alfalfa is one of the most important perennial legume forages and plays an important role in the agriculture and animal husbandry, and the ecological construction in the arid and semi-arid areas of northwest China. However, most alfalfa cultivars are difficult to maintain high forage yield in saline and arid soils since they are sensitive to salt and drought. Therefore, breeding new alfalfa cultivars with strong salt and drought tolerance is urgently needed for developing the alfalfa grass industry in arid and semi-arid regions of China. Conventional breeding method often takes a long time and is readily affected by limited genes resources within species. Efforts on generating transgenic alfalfa lines with well salt and drought tolerance using biotechnology have the important practical significance for increasing forage yield of alfalfa pastures, decreasing irrigation, and reusing salinity land in the arid and semi-arid areas of northwest China.It has been demonstrated that overexpressing the genes of tonoplast Na+/H+ antiporter and H+-pyrophosphatase (H+-PPase) can significantly improve the salt tolerance and drought resistance of plants. But those genes engaged in previous studies are mainly from model plants, crops and halophytes, and transformation was mainly performed by single gene, which might reduce the effects of these genes to improve stress resistance of plants. Thereforem, our laboratory cloned tonoplast Na+/H+ antiporter and H+-PPase genes, ZxNHX and ZxVP1-1 from a succulent xerophyte Zygophyllum xanthoxylum that is well adapted to arid environments, and constructed a bivalent-expression vectors contained above two genes. Based upon our previous work, this study co-transferred ZxNHX and ZxVP1-1 into an alfalfa cultivar "Xinjiang daye" mediating by Agrobacteriwn tumefaciens and obtained the transgenic lines co-expressing these two genes. Finally, we detected the salt and drought tolerances, and evaluated the field comprehensive phenotypes of transgenic alfalfa. The main results are as follows:1. This study firstly co-transferred tonoplast Na+/H+antiporter and H+-PPase genes from xerophyte into alfalfa and obtained 25 transgenic lines. PCR analysis showed that exogenous genes have been integrated into transgenic alfalfa genome. RT-PCR analysis indicated ZxNHX and ZxVP1-1 co-expressed in all transgenic lines. Compared with other lines, line 2,8 and 24 showed the highest expression level of both genes.2. The co-expression of ZxNHX and ZxVP1-1 enhanced salt and drought tolerance in transgenic alfalfa. In non-stress condition, the transgenic plants developed faster to larger size than wild-type plants. Under either salt or drought treatment, wild type plants exhibited growth reduction, chlorosis, biomass decrease, even die, whereas transgenic plants continued to grow well and showed higher shoot height, root length and biomass significantly than wild-type plants. The physiological analyses showed that compared with wild type plants, transgenic alfalfa accumulate more Na+, K+ and Ca2+ in their leaves and roots, maintained higher leaf relative water content and net photosynthetic rate. These results suggested that the co-expression of ZxNHX and ZxVP1-1 might increase sequestration of Na+ into vacuole, which reduced the deleterious effects of excess Na+ in the cytosol, and uptake capacity of other cations in the transgenic alfalfa. These resulted in more water retention and high photosynthetic capacity in transgenic alfalfa under salt or drought stress.3. Field experiments showed that the comprehensive phenotypes of transgenic alfalfa co-expressing ZxNHX and ZxVPl-1 were superior to wild type in either greenhouse or field. Compared with wild type plants, the transgenic alfalfa grew faster, possessed stronger photosynthetic capacity and higher biomass. |
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