Chromosome Numbers And Karyotypes Of Halogeton,Cloning Of Salt-tolerant Gene And Genetic Transformation Of Halogeton Glomeratus

Salinity is one of the major abiotic stress factors that adversely affect the growth and yield of crop plant. It is harmful to plants mainly in osmotic stress and ion stress etc,affecting the plant cell membrane structure, enzyme activity, photosynthesis and respiration organs. Especially, Salinity effected of the plant to produce reactive oxygen species,resulting in oxidative stress, so that crop growth is unbalanced, production is reduced or even death. So through agricultural measure can be Improved saline alkali land, or using genetic engineering technology to cultivate salt tolerant crop varieties to improve the adapt ability of crops.Halogeton glomeratus and Halogeton arachnoideus Moq are one of annual herbs belong to Chenopodiaceae. They mainly distributed in northwestern Gansu,xinjiang,qinghai and xizang, and a typical salt plant.in this study, we were established two species karyotype formulas and the karyotype classification, in order to provide important scientific basis on H.glomeratus genome sequencing of accurate analysis and halophytes species identification,genetic relationship, genetic diversity of germplasm resources.H.glomeratus plants there are many branches of stems on the ground, has the ability to maintain soil and water, prevent sand, salt tolerance and drought resistance. Especially, it is contains a lot of salt-tolerant genes, according to gene expression difference of the expression under the salt and alkali mixed stress, it is very likely to dig a large number of favorable genes about salt toleranceto and it is significant for cultivate salt tolerant crop varieties to improve the adaptability of crops. The main experimental results of this research are as follows:(I) Chromosome numbers and karyotypes of halogetonTo analyse the cell chromosome karyotypes of H.glomeratus and H.arachnoideus Moq.The result showed that the two kinds of plants were diploid, the indexes of cell chromosome were 18, basic chromosome number were confirmed to be x=9. The karyotype formula of two cultivars were as follows: H. glomeratus was 2n=2x=18=12m+6sm, H.arachnoideus Moq was 2n=2x=18=16m+2sm. The chromosome were mainly composed by median region chromosome(m) and submedian region chromosome(sm). Its relative length range of chromosome is 7.808%-14.888% and 7.5%-15.13%, the indexes of karyotype asymmetric coefficient of two cultivars were 61.908% and 41.662%. Respectively, their karyotype symmetries were 2A and 1B type. It can be seen that H. arachnoideus Moq belongs to relatively old or the original plant in evolutionary processes.(II) Salt-tolerant gene cloning1 With H.glomeratus c DNA template, the use of RACE technology obtained Unigene19352 and Unigene18968 sequence of 3'end, these fragments were overlapped,getting H. glomeratus 3' terminal fragment 555 bp and 608 bp, this work will lay a good foundation for later getting the full length c DNA.2 Unigene19095 of H. glomeratus was isolated by RT-PCR methods. Through analysis showed that: The full length of Unigene19095 c DNA sequence was 678 bp, contained a 388 bp 3'-UTR and a 29 bp 5'-UTR, an ORF of 261 bp which encoded a deduced protein about9.67 k Da and composed of 86 amino acid residues. Software predicted Unigene19095 encoding protein has a signal peptide, this protein with two transmembrane region,secondary structure of alpha helix, extending chain and random curl, beta angle of each accounted for 22.09%, 36.05%, 37.21%, 4.65%. Contains three phosphorylation sites, but the specific function of the gene is still need to be further studied.(III) To construct the plant expression vector CPB-Unigene19095Successful constructed expression vector CPB-Unigene19095, it was transferred into agrobacterium LBA4404, using infect inflorescence method transformed an Arabidopsis thaliana, and obtain transgenic Arabidopsis thaliana plants T1 generation. The results of PCR detection can help preliminary judging that Unigene19095 has mediated into Arabidopsis thaliana, which laid a foundation for get the homozygous transgenic plants and later transgenic offspring represent type testing and identification of gene funtion.