Influence Of BR Signaling Pathway Key Gene Expression And The Development Process Under Low Temperature Induction In Tobacco Seedling Stage

Tobacco is an important leaf used cash crop, there is a direct relation between flower bud differentiation and leaf generation; premature flower bud differentiation, can lead to the early termination of vegetative growth and reproductive growth earlier, would affect the output and quality of tobacco indirectly. Low temperature at seeding stage is one of important inducement of premature flowering. To explore the impact of low temperature at seeding stage on tobacco growth and development and the action method, we performed low temperature induction of 36 different varieties of flue-cured tobaccos during seedling stage, and observed the growth process after transplanting, which aimed at understanding the impact of seedling stage low temperature induction on different varieties of flue-cured tobaccos. According to preliminary test results of gene chip, NtBRI1 gene has higher expression levels in tobacco processed under low temperature at seeding stage than in tobacco processed under normal temperature, and the difference is more obvious over time. And a key candidate gene NtBRI1 that was closely related to low temperature at seeding stage response mechanism was cloned, furthermore, NtBSK1,NtBSK2 and NtBSK3 genes in the signal pathway were also cloned, and performed bioinformatics analysis. Using qRT-PCR strategy, the dynamic expression of NtBSK1,NtBSK2,NtBSK3, and NtFLC genes related to flower bud differentiation were analyzed in two tobacco varieties, Yunyan 85 and its multi-foliate mutant Xingyan No.1. Both were processed under low temperature(12 ℃) and normal temperature(28℃) for 10 days at 6-leaf stage. The results were as follows:(1) Low temperature at seeding stage process had double effects on the growth of most tobacco, including promoting the development process of tobacco plant and floral bud inducement. During the investigation of the growth of flue-cured tobaccos, the phenomenon was discovered that low temperature processed at seeding stage could accelerates tobacco plant development process, the leaves differentiate was faster than seedlings processed under normal temperature, and the plant was higher, the stalk was stronger with the largest leaf length and width, and the plant growed quickly. At the same time, the growing process started in advance, leaves differentiate terminated early, tobacco plant went into flower bud differentiation stage in advance, vegetative growth terminated early and entered into reproductive growth, eventually unhealthy phenomenon occured, such as insufficient leaves differentiate, thin plant, small leaves and so on. We also found that individual species was insensitive to low temperature in seedling stage, those phenomenon were not occured,such as Qinyan 201.(2) BSK is the receptor kinase of BRI1.Based on homologous cloning, three NtBSKs genes, NtBSK1, NtBSK2 and NtBSK3 in tobacco BSK gene family that participate in BR signal pathway were confirmed.With NtBSK1 ORF 1461 bp, coding 486 amino acids, NtBSK2 ORF 1479 bp, coding 492 amino acids, NtBSK3 ORF 1485 bp, coding 495 amino acids, all had a kinase structural domain PKc at N end, leucine repeated TPR structural domain at C end, excluding transmembrane region and signal peptide, all containing Ser, Thr, Tyr kinase recognition site, with strong hydrophile. This was consistent with the structural feature of the family members in arabidopsis, indicating that NtBSK1, NtBSK2 and NtBSK3 genes were all typical receptor cytoplasm kinase subfamily genes. NtBSK1 and NsBSK3 had the closest genetic relationship with NsBSK3 of homologous tobacco, they were in the same evolutionary line clade, and they had a close genetic relationship with potato and tomato which also belonged to solanaceae. And NtBSK2 has the closest genetic relationship with Arabidopsis AtBSK2.(3)Through quantitative analysis of NtBSK1, NtBSK2, NtBSK3 and NtFLC genes, we found that the relative expressions of NtBSKs increased in Xingyan 1 and Yunyan 85 after low temperature induction processing at seedling stage, with NtBSK3 gene increased most, which indicated that the BR signal transduction pathway movement was enhanced under low temperature processing, while the expression quantity of NtFLC was restrained by low temperature, thus leading to the advanced differentiation of flower bud of plants after low temperature processing. But the restrain ability had no positive correlation with the activity of BR signal transduction pathway running, which indicated that jarovization could have limited impact on BR signal transduction pathway on the regulation of tobacco plant flower bud differentiation time, and there may be another independent regulation approach.