Verification and Analysis of Cold Resistance of CcCBFc Gene from Cinnamomum camphora
WANG Jian-Ge1,2, ZHOU Chan1, LIU Yi-Lang1, DU Li1,*
1 School of Life Science and Technology, Nanyang Normal University, Nanyang 473000, China; 2 College of Landscape Architecture and Art, Henan Agricultural University College of Landscape Architecture and Art, Zhengzhou 450002, China
Abstract:CBF (CRT/DRE binding factor) gene plays an important role in ennhancing the ability of plants to resist low temperature stress. In order to verify the cold-resistance function of CcCBFc gene from Cinnamomum camphora, the mechanism of the cold-resistant effect of the CcCBFc in Arabidopsis thaliana was revealed, and the biological function of this gene in Arabidopsis against cold stress was presumed. In the early stage of this laboratory, 4 CcCBFs (CcCBFa, CcCBFb, CcCBFc, CcCBFd) were cloned, and the CcCBFc (GenBank No. KP336741) was initially verified to improve the drought resistance, salt resistance and cold resistance of the transgenic tobacco. This study was based on this, which used transgenic CcCBFc Arabidopsis and wild-type as materials to detect antioxidant enzyme activity under low temperature stress (4 ℃). Antifreeze effect of transgenic CcCBFc Arabidopsis was detected by -4 ℃ simulated freezing injury. qRT-PCR was used to detect the expression level of antioxidant gene of transgenic CcCBFc Arabidopsis at 6 d after low temperature stress, and sequenced transgenic CcCBFc Arabidopsis and wild type at transcriptional level under low temperature stress. The results showed that CcCBFc could increase the peroxidase and catalase activities of transgenic Arabidopsis, and had a positive effect on the antifreeze of transgenic Arabidopsis, and could promote the expression of antioxidant genes in transgenic Arabidopsis. Transcriptome sequencing results of the wild-type and transgenic CcCBFc Arabidopsis indicated that the function of the exogenous CcCBFc gene was related to the metabolic process, catalytic activity and binding activity, and was involved in the regulation of the endoplasmic reticulum. The synthesis of proteins and plant hormones enhanced the cold tolerance of Arabidopsis. This study provides a reference for further revealing the function of CcCBFc in the response mechanism of low temperature stress.
王建格, 周婵, 刘译朗, 杜丽. 香樟CcCBFc基因抗寒功能验证及分析[J]. 农业生物技术学报, 2021, 29(2): 268-278.
WANG Jian-Ge, ZHOU Chan, LIU Yi-Lang, DU Li. Verification and Analysis of Cold Resistance of CcCBFc Gene from Cinnamomum camphora. 农业生物技术学报, 2021, 29(2): 268-278.
[1] 蔡克桐, 沈其文, 黄志谋, 等. 2014. 茉莉酸对低温胁迫水稻幼苗的生理效应[J]. 湖北农业科学, 53(15): 3512-3515. (Cai K T, Shen Q W, Huang Z M, et al.2014. Physiological effects of jasmonate (JA) on rice seedlings under low temperature[J]. Hubei Agricultural Sciences, 53(15): 3512-3515.) [2] 李勇鹏, 张佳佳, 张力维, 等. 2016a. 香樟两个DREB1转录因子基因的分离及其对不同胁迫的响应分析[J]. 园艺学报, 44(8): 34-40. (Li Y P, Zhang J J, Zhang L W, et al.2016a. Isolation and expression profiles analysis of two DREB1 transcription factor genes under different stresses in Cinnamomum camphora[J]. Acta Horticulturae Sinica, 43(4):743-751) [3] 李勇鹏, 张力维, 张佳佳, 等. 2016b. 香樟CcCBFs基因的克隆及表达模式[J]. 东北林业大学学报, 44(08): 34-40. (Li Y P, Zhang L W, Zhang J J, et al.2016b. Cloning and expression profiling of CcCBF genes in Cinnamomum camphora[J]. Journal of Northeast Forestry University, 44(8): 34-40.) [4] 李忠光, 龚明. 2008. 愈创木酚法测定植物过氧化物酶活性的改进[J]. 植物生理学报, 44(2): 323-324. (Li Z G, Gong M .2008. Improvement of determination of plant peroxidase activity by guaiacol method[J]. Journal of Plant Physiology, 44(2): 323-324.) [5] 刘晨旭, 刘彧, 刘杰, 等. 2018. 过量表达甘菊CBF1基因提高拟南芥抗旱耐盐能力[J]. 草业科学, 35(6): 1400-1408. (Liu C X, Liu W, Liu J, et al.2018. Overexpression of Chrysanthemum lavandulifolium CBF1 gene enhances resistance to drought and salt tolerance in Arabidopsis thaliana[J]. Pratacultural Science, 35(6): 1400-1408.) [6] 滕进婧, 李梦芸, 郭纯, 等. 2018. 冷冻胁迫转金柑MLP2-1基因拟南芥的转录组测序和代谢通路[J]. 湖南农业大学学报(自然科学版), 44(04): 376-381. (Teng J Y, Li M Y, Guo C, et al., 2018. Transcriptome sequencing and metabolic pathway analysis of transgenic MLP2-1 gene in Arabidopsis under cold stress[J]. Journal of Hunan Agricultural University (Natural Science Edition), 44(04): 376-381.) [7] 唐宁. 2017. 基于芯片数据的拟南芥抗旱相关基因挖掘研究[D]. 硕士学位论文, 湖南农业大学, 导师: 张红燕, pp: 2-25. (Tang N.2017. Research on drought-related gene mining of Arabidopsis thaliana based on chip data[D]. Thesis for M.S., Hunan Agricultural University, Supervisor: Zhang H Y, pp: 2-25. ) [8] 王豪, 张波, 陆云峰, 等. 2019. 香樟新品种'御黄'[J]. 园艺学报, 46(S2): 1-2. (Wang H, Zhang B, Lu Y F, et al., 2019. A new Cinnamomum camphora cultivar 'Yuhuang'[J]. Journal of Horticulture, 46(S2): 1-2.) [9] 王洋, 胡喆, 王崇英. 2007. 拟南芥CBF/DREB途径的研究进展及其在植物基因工程中的应用[J]. 生物物理学报, 23(2): 101-108. (Wang Y, Hu W, Wang C Y, 2007. Advances of Arabidopsis thaliana CBF pathway and its implication in plant gene engineering[J]. Acta Biophysica Sinica, 23(2): 101-108. ) [10] 王建格. 2019. 转香樟CcCBFs基因拟南芥抗寒性分析及转录组测序[D]. 硕士学位论文, 南阳师范学院, 导师: 杜丽, pp: 18-37. (Wang J G .2019. Cold Resistance and Transcriptome sequencing analysis of transgenic Arabidolsis over-expressing CcCBFs[D]. Thesis for M.S., Nanyang Teachers College, Supervisor: Du L, pp: 18-37.) [11] 王羽晗, 李子豪, 李世彪, 等. 2018. 植物抗冻蛋白研究进展[J]. 生物技术通报, 34(12): 16-26. (Wang Y Z, Li Z H, Li S Z, et al.2018. Research advances on plant antifreeze Protein, 34(12): 16-26.) [12] 余琼芳. 2018. 乌菜CBF1基因转化番茄与拟南芥及其功能验证[D]. 硕士学位论文, 安徽农业大学, 导师: 刘童光, pp. 19-28. (Yu Q F.2018. Transformation of CBF1 into tomato and Arabidopsis thaliana and its function verification[D]. Thesis for M.S., Anhui Agricultural University, Supervisor: Liu T G, Anhui Agricultural University, pp. 19-28.) [13] 于波. 2017. 转StCBF1与转ScCBF1基因拟南芥抗冻性与冷驯化能力研究[D]. 硕士学位论文, 山东农业大学, 导师: 杨兴洪, pp: 2-15. (Yu B.2017. Study on the mechanisms of freezing tolerance and cold acclimation capacity between StCBF1-tansgenic and ScCBF1-tansgenic Arabidopsis[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Yang X H, pp: 2-15.) [14] 于欣鑫, 高晋, 君李勇, 等. 2014. flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化[J]. 中国生物工程杂志, 34(5): 30-38. (Yu X X, Gao J, Jun L Y, et al.2014. Transcriptome analysis of Arabidopsis thaliana and changes of glucosinolates metabolism pathway induced by Flg22[J]. Chinese Biotechnology, 34(5): 30-38.) [15] 赵滢, 杨义明, 范书田, 等. 2017. 木本植物抗寒性的环境调控及响应机制研究进展[J]. 分子植物育种, 14(2): 750-756. (Zhao W, Yang Y M, Fan S T, et al.2017. Research progress on environmental regulation and response mechanism of cold resistance in woody plants[J]. Molecular Plant Breeding, 14(2): 750-756. ) [16] 张力维, 李勇鹏, 姚瑶, 等. 2015. 香樟延伸因子EF1a基因片段的克隆及表达分析[J]. 中南林业科技大学学报, (5): 122-128. (Zhang L W, Li Y P, Yao Y, et al. 2015. Cloning and expression analysis of EF1a gene fragment of elongation factor from Cinnamomum camphora[J]. Journal of Central South University of Forestry and Technology, (5): 122-128.) [17] 张兰, 滕珂, 肖国增, 等. 2016. 日本结缕草ZjADH基因对拟南芥的转化及其耐寒性分析[J]. 草业学报, 25(11): 43-49. (Zhang L, Teng B, Xiao G Z, et al.2016. Transformation of ZjADH gene into Arabidopsis thaliana and cold-tolerance analysis of transgenic plants[J]. Acta Prataculturae Sinica, 25(11): 43-49.) [18] 张增艳, 陈洋, 邵艳军. 2009. 植物激素调控植物防御反应的研究进展[J]. 作物杂志, (6): 13-17. (Zhang Z Y, Chen Yang, Shao Y J. 2009. Review of research advances on phytohormones regulating plant defenser esponses[J]. Journal of Crops, (6): 13-17.) [19] 周婵, 王建格, 张佳佳, 等. 2019. 过表达香樟CcCBFs提高转基因拟南芥抗寒能力[J]. 东北林业大学学报, 47(3): 56-61. (Zhou C, Wang J G, Zhang J J, et al.2019. Overexpression of CcCBFs from Cinnamomum camphora in enhancing ability of transgenic Arabidopsis thaliana[J]. Journal of Northeast Forestry University, 47(3): 56-61.) [20] Cheng Z H, Lei N, Li S X, et al.2019. The regulatory effects of MeTCP4 on cold stress tolerance in Arabidopsis thaliana: A transcriptome analysis[J]. Plant Physiology and Biochemistry, 138(02): 9-16. [21] Chinnusamy, Viswanathan, Zhu J H, et al.2006. Gene regulation during cold acclimation in plants[J]. Physiol Plantarum, 126(1): 52-61. [22] Buskirk H A V, Thomashow M F.2006. Arabidopsis transcription factors regulating cold acclimation[J]. Physiol Plantarum, 126(1): 72-80. [23] Steven H S, Gary J L.2011. Redox-based protein modifications: The missing link in plant immune signaling[J]. Current Opinion in Plant Biology, 14(4): 358-364. [24] Kazan K.2015. Diverse roles of jasmonates and ethylene in abiotic stress tolerance[J]. Trends in Plant Science, 20(4): 219-29.