Effect of RD29A Promoter on the Stress Resistance of Transgenic Tobacco with SikCDPK1 Gene from Saussurea involucrata

doi:10.13560/j.cnki.biotech.bull.1985.2023-0150

Abstract

Abstract:

This work is to explore the effect of the stress-induced promoter RD29A on the stress tolerance of transgenic tobacco with SikCDPK1 gene from Saussurea involucrata, thus which may lay the foundation for SikCDPK1 gene to play a more effective role when the plant encounters low temperature and drought. Gene recombination technology was used to construct the plant expression vector of SikCDPK1 gene driven by RD29A promoter, and genetic transformation into tobacco by agrobacterium-mediated method, and the phenotype, chlorophyll content, MDA content, POD activity, SOD activity and relative conductivity of RD29A∷SikCDPK1 transgenic tobacco and 35S∷SikCDPK1 transgenic tobacco and non-GMO tobacco were observed, measured and compared after low temperature and drought treatment. As results, the growth status of RD29A∷SikCDPK1 transgenic tobacco was better than that of 35S∷SikCDPK1 transgenic tobacco, and better than that of non-GMO tobacco after drought and low temperature stress. At the same time, the chlorophyll content, POD activity and SOD activity of RD29A∷SikCDPK1 transgenic tobacco were significantly higher than those of 35S∷SikCDPK1 transgenic tobacco, which was very significantly higher than that of non-GMO tobacco. However, the relative conductivity and MDA content of RD29A∷SikCDPK1 transgenic tobacco were significantly lower than 35S∷SikCDPK1 transgenic tobacco, which was very significantly lower than that of non-GMO tobacco. In sum, the promoter RD29A could show stronger drought and low temperature tolerance by slowing down the degradation rate of chlorophyll, increasing enzyme activity of antioxidant system and reducing membrane permeability.

Key words: SikCDPK1 gene from Saussurea involucrata, RD29A promoter, GMO, drought, low temperature, tolerance

LIU Yu-ling, WANG Meng-yao, SUN Qi, MA Li-hua, ZHU Xin-xia. Effect of RD29A Promoter on the Stress Resistance of Transgenic Tobacco with SikCDPK1 Gene from Saussurea involucrata[J]. Biotechnology Bulletin, 2023, 39(9): 168-175.

Figures/Tables 5

Table 1 Primers information

引物Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
RD29A-F	GAATTCCGACTCAAAACAAACTTACGAA	启动子克隆Promoter cloing
RD29A-R	CCCGGGAATCAAACCCTTTATTCCTGA	启动子克隆Promoter cloing
SikCDPK1-F	GGATCCATGGGGAATACTTGTGTTGGAC	基因鉴定 Gene identification
SikCDPK1-R	GTCGACCCGTCGATACCGGAAAAAAC	基因鉴定 Gene identification

Fig. 1 Double enzyme digestion of RD29A∷SikCDPK1 recombinant plasmid A: Recombinant plasmids was digested with double enzymes（M: Marker III.1: Plasmid control. 2: Digestion products）. B: Recombinant plasmids were digested with double enzymes（M: Trans 5 K DNA marker. 1-2: Digestion products.3: Plasmid control）

Fig. 2 Identification of transgenic tobacco by PCR A: PCR identification of 35S∷SikCDPK1 transgenic tobacco（M: DL2000 DNA marker. 1-17: Transgenic plant）. B: PCR detection of RD29A∷SikCDPK1 transgenic tobacco SikCDPK1 gene（M: DL2000 DNA marker. 1-7: Detection of SikCDPK1 gene PCR. 8-16: WT PCR control）. C: PCR detection of RD29A∷SikCDPK1 transgenic tobacco RD29A promoter（M: Marker III. 1-7: RD29A promoter PCR detection. 8: WT PCR control）

Fig. 3 Phenotypic characteristics of WT tobacco and transgenic tobacco under stress A, C: Normally growing tobacco. B: Withheld watering 30 d. D: Low temperature treatment of tobacco after 48 h. Line 1, Line 2: Two different tobacco lines

Fig. 4 Physiological indexes of non-transgenic tobacco and transgenic tobacco determined before and after stress * and * * showed significant correlations at 0.05 and 0.01 levels, respectively

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