启动子RD29A对转雪莲SikCDPK1基因烟草抗逆性的影响

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

摘要/Abstract

摘要：

探究逆境诱导启动子RD29A对转雪莲SikCDPK1基因烟草抗逆性的影响，为SikCDPK1基因在植物遭遇低温、干旱时更好发挥作用奠定基础。采用基因重组技术构建RD29A启动子驱动SikCDPK1基因的植物表达载体，通过农杆菌介导法遗传转化烟草，分别观察、测定和比较分析低温、干旱处理后，RD29A∷SikCDPK1转基因烟草、35S∷SikCDPK1转基因烟草和非转基因烟草的表型、POD活性、SOD活性、叶绿素含量、MDA含量和相对电导率的差异。结果显示，干旱和低温胁迫后，RD29A∷SikCDPK1转基因烟草的生长状况优于35S∷SikCDPK1转基因烟草，更优于非转基因烟草；同时，RD29A∷SikCDPK1转基因烟草的POD活性、SOD活性、叶绿素含量显著高于35S∷SikCDPK1转基因烟草，极显著高于非转基因烟草；但MDA含量与相对电导率显著低于35S∷SikCDPK1转基因烟草，极显著低于非转基因烟草。表明启动子RD29A可通过减缓叶绿素降解速率、提高抗氧化系统酶活性、减小膜通透性，使转基因烟草表现出更强的干旱、低温耐受性。

关键词: 雪莲SikCDPK1基因, RD29A启动子, 转基因, 干旱, 低温, 耐受性

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

刘玉玲, 王梦瑶, 孙琦, 马利花, 朱新霞. 启动子RD29A对转雪莲SikCDPK1基因烟草抗逆性的影响[J]. 生物技术通报, 2023, 39(9): 168-175.

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.

图/表 5

表1 引物序列信息

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

图1 RD29A∷SikCDPK1重组质粒双酶切鉴定 A: 重组质粒双酶切（M：Marker III；1：质粒对照； 2：酶切产物）；B: 重组质粒双酶切（M：Trans 5 K DNA marker；1-2：酶切产物；3：质粒对照）

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）

图2 转基因烟草PCR鉴定 A：35S∷SikCDPK1转基因烟草PCR鉴定（M：DL2000 DNA marker；1-17：转基因植株）；B：RD29A∷SikCDPK1转基因烟草SikCDPK1基因PCR检测（M：DL2000 DNA marker；1-7：SikCDPK1基因PCR检测；8-16：非转基因烟草PCR对照）；C：RD29A∷SikCDPK1转基因烟草RD29A启动子PCR检测（M：Marker III；1-7：RD29A启动子PCR检测；8：非转基因烟草PCR对照）

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）

图3 非转基因烟草与转基因烟草逆境胁迫前后表型变化 A，C：正常生长的烟草；B：干旱30 d的烟草；D：低温处理48 h 后的烟草； Line 1，Line 2：不同株系

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

图4 非转基因烟草与转基因烟草胁迫处理前后生理指标测定 *与**分别表示相关性达显著（P<0.05）和极显著（P <0.01）水平

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

参考文献 26

[1]	Li YY, Zhang HX, Liang SB, et al. Identification of CDPK gene family in Solanum habrochaites and its function analysis under stress[J]. Int J Mol Sci, 2022, 23(8): 4227. doi: 10.3390/ijms23084227 URL
[2]	Bi ZZ, Wang YH, Li PC, et al. Evolution and expression analysis of CDPK genes under drought stress in two varieties of potato[J]. Biotechnol Lett, 2021, 43(2): 511-521. doi: 10.1007/s10529-020-03037-2
[3]	李建鑫. 番茄钙依赖蛋白激酶CPK28在植物响应高温胁迫中的功能及机制研究[D]. 杭州: 浙江大学, 2021.
	Li JX. The function and mechanism of calcium-dependent protein kinases CPK28 in tomato response to heat stress[D]. Hangzhou: Zhejiang University, 2021.
[4]	朱丽萍, 于壮, 邹翠霞, 等. 植物逆境相关启动子及功能[J]. 遗传, 2010, 32(3): 229-234.
	Zhu LP, Yu Z, Zou CX, et al. Plant stress-inducible promoters and their function[J]. Hereditas, 2010, 32(3): 229-234.
[5]	Orbović V, Ali Ravanfar S, Acanda Y, et al. Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp[J]. Transgenic Res, 2021, 30(5): 687-699. doi: 10.1007/s11248-021-00260-z
[6]	Bihmidine S, Lin JS, Stone JM, et al. Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress[J]. Planta, 2013, 237(1): 55-64. doi: 10.1007/s00425-012-1740-9 pmid: 22983672
[7]	聂利珍, 于肖夏, 李国婧, 等. Rd29A启动子驱动AtCDPK1基因转化马铃薯的研究[J]. 中国生物工程杂志, 2015, 35(11): 13-22.
	Nie LZ, Yu XX, Li GJ, et al. Study on transgenic potato contained AtCDPK1 gene drived by Rd29A promoter[J]. China Biotechnol, 2015, 35(11): 13-22.
[8]	Rai GK, Rai NP, Rathaur S, et al. Expression of RD29A∷AtDRE-B1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants[J]. Plant Physiol Biochem, 2013, 69: 90-100. doi: 10.1016/j.plaphy.2013.05.002 URL
[9]	Estrada-Melo AC, Chao, Reid MS, et al. Overexpression of an ABA biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia[J]. Hortic Res, 2015, 2: 15013. doi: 10.1038/hortres.2015.13
[10]	Das A, Basu PS, Kumar M, et al. Transgenic chickpea(Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit[J]. BMC Plant Biol, 2021, 21(1): 39. doi: 10.1186/s12870-020-02815-4
[11]	Kang HH, Zhang M, Zhou SM, et al. Overexpression of wheat ubiquitin gene, Ta-Ub2, improves abiotic stress tolerance of Brachypodium distachyon[J]. Plant Sci, 2016, 248: 102-115. doi: 10.1016/j.plantsci.2016.04.015 URL
[12]	Gu XB, Chen YH, Gao ZH, et al. Transcription factors and anthocyanin genes related to low-temperature tolerance in RD29A∷RdreB1BI transgenic strawberry[J]. Plant Physiol Biochem, 2015, 89: 31-43. doi: 10.1016/j.plaphy.2015.02.004 URL
[13]	田晓涵, 庞学兵, 祝建波, 等. 过表达天山雪莲SikCDPK1基因提高转基因烟草耐低温能力的机制初探[J]. 中国烟草学报, 2016, 22(6): 98-103.
	Tian XH, Pang XB, Zhu JB, et al. Effect of over-expression of Saussurea involucrata SikCDPK1 gene on improving cold tolerance in transgenic tobacco[J]. Acta Tabacaria Sin, 2016, 22(6): 98-103.
[14]	王金胜, 吕淑霞, 张宁. 基础生物化学[M]. 2版. 北京: 中国农业出版社, 2021.
	Wang JS, Lv SX, Zhang N. Fundamental biochemistry[M]. 2nd ed. Beijing: China Agriculture Press, 2021.
[15]	Zhang HM, Zhu JH, Gong ZZ, et al. Abiotic stress responses in plants[J]. Nat Rev Genet, 2022, 23(2): 104-119. doi: 10.1038/s41576-021-00413-0
[16]	Asano T, Hayashi N, Kikuchi S, et al. CDPK-mediated abiotic stress signaling[J]. Plant Signal Behav, 2012, 7(7): 817-821. doi: 10.4161/psb.20351 pmid: 22751324
[17]	McAinsh MR, Pittman JK. Shaping the calcium signature[J]. New Phytol, 2009, 181(2): 275-294. doi: 10.1111/j.1469-8137.2008.02682.x pmid: 19121028
[18]	Shi SJ, Li SG, Asim M, et al. The Arabidopsis calcium-dependent protein kinases(CDPKs)and their roles in plant growth regulation and abiotic stress responses[J]. Int J Mol Sci, 2018, 19(7): 1900. doi: 10.3390/ijms19071900 URL
[19]	Boudsocq M, Willmann MR, McCormack M, et al. Differential innate immune signalling via Ca²⁺ sensor protein kinases[J]. Nature, 2010, 464(7287): 418-422. doi: 10.1038/nature08794
[20]	Zou JJ, Wei FJ, Wang C, et al. Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca²⁺-mediated stomatal regulation in response to drought stress[J]. Plant Physiol, 2010, 154(3): 1232-1243. doi: 10.1104/pp.110.157545 URL
[21]	Ampo S, Baldrich P, Messeguer J, et al. Overexpression of a calcium-dependent protein kinase confers salt and drought tolerance in rice by preventing membrane lipid peroxidation[J]. Plant Physiol, 2014, 165(2): 688-704. pmid: 24784760
[22]	Kiselev KV, Aleynova OA, Ogneva ZV, et al. 35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress[J]. Mol Biol Rep, 2021, 48(3): 2235-2241. doi: 10.1007/s11033-021-06235-x
[23]	Zhou SM, Sun XD, Yin SH, et al. The role of the F-box gene TaFBA1 from wheat(Triticum aestivum L.) in drought tolerance[J]. Plant Physiol Biochem, 2014, 84: 213-223. doi: 10.1016/j.plaphy.2014.09.017 URL
[24]	Wei T, Deng KJ, Zhang QX, et al. Modulating AtDREB1C expression improves drought tolerance in Salvia miltiorrhiza[J]. Front Plant Sci, 2017, 8: 52.
[25]	Pino MT, Skinner JS, Park EJ, et al. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield[J]. Plant Biotechnol J, 2007, 5(5): 591-604. doi: 10.1111/pbi.2007.5.issue-5 URL
[26]	Li F, Han YY, Feng YN, et al. Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development[J]. J Biotechnol, 2013, 163(3): 281-291. doi: 10.1016/j.jbiotec.2012.11.008 pmid: 23183383