Cloning And Sequencing Analysis Of Cold-related Resistance Gene In Wheat

Injury by low temperture not only restricts the plant range of crop, but also causes yield reduction. the loss of crops caused by low temperature is up to hundreds of billions RMB in the world every year.Studying and improving the cold tolerance of plants play a significant role in the theoretical and actual aspects. For clarify the relationship between cold induced gene and cold resistance of wheat seedlings, in this research, we selected260cultivars from the different wheat growing zones in China and some of introduced varieties to identify the seedlings cold resistance, and STS detection of cold-regulated gene. Based on the mRNA sequence of24cold-regulated gene from NCBI, several pairs of primers were designed.260wheat cultivars were used for PCR.the results indicate: Wrab19（GenBank Accession Number：AF139915）、Wcs19（GenBank Accession Number： L13437）、Wrab17(GenBank Accession Number：AF255053) three cold-regulated gene associated with cold resistance of Wheat.1. Cold resistance identification in laboratory indicate: cold resistance of260cultivars distribute broadly,very obvious difference between varieties, all cultivars the range of cold resistance was0.0~1.0,phenotype difference is more significantly.2.Amplification to Wrab19gene use the primer,polymorphism was very appearance.amplified two fragment of690bp and1200bp in133cultivars one fragment of690bp in127cultivars.Select4cultivars of different cold resistance was sequencing on random,the name of cultivars was Serra、02Y151、s u9908、C P93-18-1-1-1-5，th e sequence of front two cultivars was no difference with primary alignment,but the cultivars that last two was more33bp fragment in222bp,speculate may be allele gene.3．Amplification to Wcs19gene use the primer to260cultivars,the results showed that wcs19gene without difference in DNA level among260wheat cultivars, cold resistance of seedlings may related to wcs19gene expression.Further sequence analysis demonstrated that the DNA sequence of wcs19gene has an121-122bp intron，which contains an SNP, namely, at the+9bp has one insertion and deletion of G base in the intron sequence. Further study is to needed on the relationship between the SNP of intron and cold resistance of wheat seedlings.4. Amplification to Wrab17gene use the primer that specific is better, practical amplificate the fragment of610bp but predicte target was only468bp, polymorphic was not exist between varieties.Wrab17gene without difference in DNA level among260wheat cultivars, Select2cultivars of different cold resistance was sequencing on random, DH5037andDH6197,cold resistance was0.86and0.57separately.sequencing results showed: two sequence has an142bp intron,in addition,the intron boundary sequence was mutate of DH6197cultivar,from G to T,that the primary cause of cold resisitant was decined.5. The rest21gene detected to260cultivar proved that cold regulated gene ubiquity in wheat.