| The young shoots of tea trees are good traits of tea varieties.The trichome is rich in amino acids,polyphenols,caffeine,flavonoids,and its biochemical properties give more health benefits and a variety of taste,such as bitterness,astringency,and sweetness.At present,studies on trichome growth of tea plants in and abroad have stayed in the biochemical characteristics and its resistance.There is no research on the related genes of tea tree trichome,mainly in the plants of Arabidopsis thaliana,Malus domestica so on.To explore the molecular mechanism regulating the formation and development of tea tree pubescence is of great significance for the development of tea tree breeding.In this study,the previous transcriptome data was used to obtain the WD40 transcription factor(CsTTG1),and the transcription factor CsTTG1 was obtained for analysis and functional identification.To identify the linear relationship between the growth and development of CsTTG1 and trichome,we constructed the overexpression vector pBI121-CsTTG1 to transform Arabidopsis thaliana and tobacco model plants.Gene function verification yields the following results:(1)The full-length of gene CsTTG1 is 1380 bp,with 9 open reading franes when the longest length is 1029 bp.Bioinformatics Analysis of the Transcription Factor TTG1 in the Tea Tree,the phylogenetic tree was constructed by placing the TTG1 gene in 12 species including Arabidopsis thaliana,Malus domestica,Brassica napus so on and together with the transcription factor TTG1 in the tea plant.We found that it is highly homologous with NtTTG1,suggesting that it may be involved in regulating the growth and development of the epidermis as the same as the homologous gene NtTTG1 in tabacco.(2)The expression characteristics of transcription factor CsTTG1 were analyzed by qRT-PCR.The results showed that the expression level of CsTTG1 was higher in the more tea-grown tea lines,and the expression level of CsTTG1 was lower in the hairless lines.The expression of transcription factor CsTTG1 showed a linear relationship at different growth sites in the tea tree line.CsTTG1 had the highest expression in the terminal bud and the old leaf had the lowest expression.(3)The target gene CsTTG1 was recombined with the vector plasmid pBI121.Through colony validation,resistance screening and validation,the overexpression vector pBI121-CsTTG1 of tea plant CsTTG1 gene was successfully constructed and transduced in Agrobacterium GV1301.(4)Under the mediation of Agrobacterium GV1301,the overexpression vector pBI121-CsTTG1 was transformed into Arabidopsis thaliana in vitro through the method of "Floating flower".The transgenic Arabidopsis thaliana plants were obtained by rooting and transplanting in the resistant medium.The transgenic Arabidopsis plants were extracted with DNA and subjected to PCR.At the same time,fluorescence quantitative PCR analysis was performed.It was found that the positive expression level of CsTTG1 gene in the positive plants was higher than that in the blank control group.The CsTTG1 phenotype was observed with a stereomicroscope.The results showed that the number of epidermal hairs increased compared with the blank control group,and the degree of hair growth was positively correlated with the increase of the transcription level of CsTTG1,indicating that CsTTG1 is regulating hair formation.(5)Under the mediation of Agrobacterium GV1301,the overexpression vector pBI121-CsTTG1 was transferred into sterile tobacco,screened,induced to differentiate,and rooted through a series of resistant media.Finally,the positive tobacco plants were obtained by transplantation.Using RT-PCR analysis,it was found that CsTTG1 was highly expressed in 2 transgenic tabaccos,while there was no signficant change in epidermal hair.Due to differences in apparent individuals,it was unable to explain whether overexpression of CsTTG1 increased tobacco epidermal hair number.whether there were other genes involved would better control of the growth and development of hair needs futher study. |
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