Studies On High Throughput Screening Technology For Plant Salt-tolerance Related Genes

There are 6.7 million hectares of salinized arable land,accounting for 10%of the total arable land in China,in addition,there are 2.17 million hectares of marine beach,some of them are reserve land for agriculture.It is of great significance for increasing food crop production in China to increase crop yield and improve quality on salinized soil,develop new crop cultivars highly tolerant to salinity as pioneer crops for mudflat development and utilization.Rice is the staple food crop in China,especially suitable for planting on mudflat,however,the salt tolerance limit of the rice is 0.06%-0.1%.Efforts have been be making to improve the salt tolerance of rice by conventional breeding or molecular breeding all the while,and considerable progress has been made.However,It is still an important goal to further improve the salt tolerance of rice via gene introgression or transgene to introduce exogenous genes from the wild halophytes.The common way to create salt-tolerant transgenic plants is to select a few so-called "salt-tolerant genes" based on our knowledge of the salt tolerance mechanism of plant to verify their function in model plant,e.g.Arabidopsis at first,and then,select those with certain functions to introduce into target crops.It usually takes a couple of years to clarify gene function,but it always happened that the gene function does not match the expected or there is no obvious phenotype,sometimes the same gene exhibits different phenotypes in crop compared to those in model plants.In order to screen and utilize salt-tolerant gene resources in wild plants for improvement of rice and other crops rapidly and efficiently,in the present study,a novel rice transgenic method by pollinating the germinated pollen infected by Agrobacterium was established firstly,and a salt-induced cDNA library of Spartina anglica Hubb indusalt constructed using Gateway(?)technology was transferred into Agrobacterium,and was further transferred into BY-2 cells by Agrobacterium infection,and the resulting BY-2 cells were consequently screened for salt tolerance.A large-scaled salt tolerance transgenic rice screening technology platform is preliminarily established.The main results are as follows:1.Investigation of a novel transgenic methods by pollination Agrobacterium-infected pollen for rice:The cotton GhMAC3 gene was cloned from a GhMAC3 transgenic Arabidopsis by PCR and constructed to the plant expression vector pMDC83 by using Gateway(?)method.The agrobacterium transformed with recombinant pMDC83-GhMAC3 was sused to infect the rice pollen cultured in vitro,and the pollen was subsequently pollinated onto the stigma of rice var.9311,and the transgenic T0 seeds were obtained.100 T0 rice seeds were randomly picked out for germinating,the marker gene of Hpt and the target gene of GhMAC3 in each seedlings were detected by PCR respectively.Hpt was detected in 8 plants,and GhMAC3 was detected in 22 plants.The gene GhMAC3 amplified from the transgenic rice seedling by PCR was further cloned and sequenced,the result showed that the insertion sequence was same as that of cotton GhMAC3.2.Inspection method of rice hygromycin resistance:The non transgenic seeds of rice var.9311 were treated with 50 mg/L,100 mg/L,150 mg/L,167 mg/L,and 200 mg/L of hygromycin respectively,and were cultured for 2 days until sprouting.Germination and root growth of rice seeds were inhibited as the concentrations of hygromycin increased,when the concentration reached 167 mg/L,root growth was significantly blocked although the shoot sheaths normally elongated.Therefore,167 mg/L is the most suitable for screening rice hygromycin resistance.3.Preliminary screening of salt-tolerant genes from Spartina anglica Hubb indusalt:The salt-induced cDNA library of Spartina anglica Hubb indusalt constructed by Gateway(?)was transformed into tobacco BY-2 cells through Agrobacterium infection,the transformed cells were screened with 25 mmol/L hygromycin coupled with and 50 mmol/L,100 mmol/L and 200 mmol/L NaCl respectively.A number of BY-2 cell clones that can tolerate 50 mmol/L or 100 mmol/L NaCl were obtained.108 big clones that can grow relatively fast in NaCl were picked out for subculture and gene identification.