Cloning And Expression Analysis Of The Cold Resistance Related Genes From Tea Plant [Camellia Sinensis(L) O.Kuntze]

Cold is a typical environmental stress factor that limits the geographical distribution and growing season of many plant species, and it often adversely affects crop quality and productivity. However, the response of plants to low temperature stress response is not entirely passive, but is a process of active. Tea paint(Camellia sinensis) originated in tropic and subtropical regions,so that it likes warm and welsh climes and has poor tolerance to low temperature.It's important to screen and identify genes associated with cold stress, through researching gene expression of tea in the low temperature. Then cold tolerance gene was cloned from identified genes for further revealing the molecular mechanism against freezing. The results used to early identify cold tolerance of tea germplasm and genetic stability of cold resistant, which to help cold resistant breeding of tea paint, had an extremely important theoretical and practical value.In this study, Yunnan large-leaved cv. 73-11 and small-medium leaved cv. Shuchazhao as the test material. The cold regulated genes were screened from both Yunnan large-leaved cv. 73-11 and small-medium leaved cv. Shuchazhao under low temperature stress for 4 days using cDNA-AFLP. 41TDFs were obtained, including 38 TDFs from d cv. 73-11, 18 TDFs from cv.Shuchazao, and16 TDFs shared by the two varieties. According to blasting results, these TDFs are classified into four groups: 1)signal transduction genes; 2)Transcription factors; 3)Low-temperature related metabolism genes; 4)genes for protein related to stress. In addition, there were some genes for predicted protein and unknown protein. It had been shown that response of tea plant to low-temperature stress is very complex with many genes of several metabolism involved, and cv. 73-11 is more sensitive to the low temperature stress than cv.Shuchazao.The full length cDNA of differential expression fragment kh1 what was induced by low temperature was 1329 bp by using RACE, the open reading frame coding 361 amino acid residues included two conserved domains AP2, B3 and had high homology with RAV protein of many plants. So, it deduced that the gene belonged to RAV subfamily of AP2/EREBP family and was named tea plant RAV gene(GenBank accession number: GQ227992). qRT-PCR results indicated that CsRAV was induced by cold, ethylene and NaCl, and maximum relative expression was 5.8 , 10.0 and 1.9 times higher than before treatment, respectively. CsRAV gene expression level was closed in leave, bud and stem, and lower in flower and root, not detected in seed. It shows the expression of the CsRAV might be strictly controlled in different organs and plays an important role during abiotic stress in tea plant.At the same time, the full length cDNA of differential expression fragment kh11 was cloned by using RACE, the open reading frame was 636 bp and coded 212 amino acid residues and had a conserved AP2/ERF domain from 108 to 166. Sequence and structure analysis showed that it had high sequence homology with ERF transcription factors of many plants. So, it confirmed that the gene was the encoding gene of the ERF transcription factor of tea plant (GeneBank accession number: GU393024). qRT-PCR analysis showed that ERF gene were up-regulated by cold, ethylene, dehydration, NaCl and the maximum expression were 121.1, 22.6, 2.6, 2.2 times higher than before treatment, respectively. Expression Characteristics in different tissues showed that the expression of the gene was maximum in mature leaves, the next were bud, root and stem, but it was very low in flower and fruit. It deduced that ERF gene was very important in response to abiotic stress and was strictly controlled in expression of different tissues.