Functional Analysis of SlMYB96 Gene in Tomato Under Cold Stress

doi:10.13560/j.cnki.biotech.bull.1985.2022-1051

Abstract

Abstract:

This study is to discuss the function of SlMYB96 in tomato under cold resistance, aiming to provide theoretical basis for the molecular mechanism of cold resistance and breeding of tomato under cold resistance. The SlMYB96 gene was cloned using tomato cDNA as the template. Then the physical and chemical properties of the gene were analyzed by bioinformatic software, and the expression features of SlMYB96 and its role in cold resistance in tomato was studies by real-time quantitative fluorescence（RT-qPCR）technology and virus-induced gene silencing（VIGS）technology. The results showed that SlMYB96 was expressed in the roots, stems, leaves, flowers, and fruit of tomato, with the highest expression in the flowers. And the expression of SlMYB96 increased with increasing time of 4℃ cold treatment, where expression reached the maximum at three hour of low temperature treatment. With the help of VIGS the SlMYB96 gene was silenced. Three different types of tomato plants in wild-type group（WT）, empty load group（CK）and gene transient silencing group（pTRV-MYB96）were treated with low temperature. And the appearance traits showed that plants in the transient gene silencing group（pTRV-MYB96）after cold treatment at 4℃ for five days, presented more obvious cold damage symptoms compared with the wild-type group（WT）and the empty-load group（CK）. The identification results of the physiological level showed that when tomato seedlings were treated with 4℃ at low temperature for five days, the content of chlorophyll, malondialdehyde, soluble protein and superoxide dismutase activity of the gene transient silencing group（pTRV-MYB96）plants were significantly lower, while the activity of soluble sugar, relative conductivity, free proline content, catalase and peroxidase increased. When tomato seedlings treated under cold 4℃ for five days, chlorophyll, malondialdehyde, soluble protein content and superoxide dismutase activity were significantly lower in gene transient silent group（pTRV-MYB96）, while soluble sugar, relative conductivity, free proline content and catalase and peroxidase activity increased, indicating that gene transient silent group（pTRV-MYB96）plants had lower cold resistance compared with wild-type group（WT）and empty load group（CK）. It is confirmed that SlMYB96 gene can respond to cold stress and reduce cold resistance after silencing.

Key words: tomato, SlMYB96, gene function analysis, cold hardiness

HU Ming-yue, YANG Yu, GUO Yang-dong, ZHANG Xi-chun. Functional Analysis of SlMYB96 Gene in Tomato Under Cold Stress[J]. Biotechnology Bulletin, 2023, 39(4): 236-245.

Figures/Tables 8

Table 1 List of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
MYB96-F	ATGGGTAGAGTCCCTTGTTGTG
MYB96-R	CTAGAAAAGTTGTGGAGTTTCATCAAAAGATATCA
Y96-F	TGCAGGCTAAGATGGACAAACTACC
Y96-R	CTGTTCTCTCTGGCAGATACGAAGC
Actin-F	TTGCTGACCGTATGAGCAAG
Actin-R	GGACAATGGATGGACCAGAC
V96-F	TCTTCATGTGATGGGACTCCAAA
V96-R	CTAGAAAAGTTGTGGAGTTTCATCAAAAG
+V96-F	AAGGTTACCGAATTCTCTAGATCTTCATGTGATGG- GACTCCAA
+V96-R	TGTCTTCGGGACATGCCCGGGCTAGAAAAGTTGT- GGAGTTTCATCAAA

Fig. 1 PCR amplification of SlMYB96 M: BM2000 DNA marker. 1-3: Three repeats

Fig. 2 Sequence analysis of SlMYB96 A: Hydrophilicity analysis of SlMYB96 protein. B: Conserved domain analysis of SlMYB96. C: Secondary structure of SlMYB96 protein. D: Tertiary structure of SlMYB96 protein

Fig. 3 Expression pattern analysis of SlMYB96 A: Expression pattern analysis of SlMYB96 in different tissues. B: Expression pattern analysis of SlMYB96 in roots, stems and leaves under low temperature stress. Different lowercase letters indicate significant differences in at P=0.05. The same below

Fig. 4 Vector construction of pTRV2-MYB96 A: Enzyme digesting verification of pTRV2-MYB96 vector. B: pTRV2-MYB96 vector sequence alignment. M: BM2000 DNA marker.1-3: Three repeats

Fig. 5 TRV virus detection M: BM2000 DNA marker. 1-2: WT. 3-6, 8, 9: TRV positive. 7, 10: TRV negative

Fig. 6 Phenotypic observation of tomato plants silenced by VIGS A-C: 25℃ wild group, no load group, instantaneous silent plants. D-F: After treatment at 4℃ for 5 d, the wild group, the no-load group and the instantaneous silent plants were isolated

Fig. 7 Determination of physiological and biochemical indexes of tomato plants under low temperature stress A: Chlorophyll content in leaves. B: Blade relative conductivity in leaves. C: Malondialdehyde content in leaves. D: Determination of free proline content in leaves. E: Soluble sugar content in leaves. F: Soluble protein in leaves. G: Catalase activity in leaves. H: Superoxide dismutase activity in leaves. I: Peroxidase activity in leaves

References 36

[1]	施应宣. 园艺植物冷害和抗冷性分析[J]. 农业开发与装备, 2020(3): 108, 118.
	Shi YX. Analysis of cold damage and cold resistance of horticultural plants[J]. Agric Dev ＆ Equip, 2020(3): 108, 118.
[2]	彭鹏. 早春大棚优质番茄品种的综合评价及耐寒性鉴定[D]. 郑州: 河南农业大学, 2019.
	Peng P. Comprehensive evaluation and cold tolerance identification of high quality tomato varieties in early spring greenhouse[D]. Zhengzhou: Henan Agricultural University, 2019.
[3]	Albert NW, Griffiths AG, Cousins GR, et al. Anthocyanin leaf markings are regulated by a family of R2R3-MYB genes in the genus Trifolium[J]. New Phytol, 2015, 205(2): 882-893. doi: 10.1111/nph.13100 pmid: 25329638
[4]	吴小亲. SlMYB64参与番茄植株生长及花粉萌发的初步研究[D]. 泰安: 山东农业大学, 2016.
	Wu XQ. Preliminary study on SlMYB64 involved in plant grouth and pollen germination in tomato[D]. Tai'an: Shandong Agricultural University, 2016.
[5]	陈静. 番茄SlMYB1R-1基因的克隆和功能研究[D]. 重庆: 重庆大学, 2017.
	Chen J. Cloning and functional analysis of SlMYB1R-1 gene in tomato[D]. Chongqing: Chongqing University, 2017.
[6]	陈丽琛. 番茄SlMYB102基因的克隆及耐盐功能的初步鉴定[D]. 泰安: 山东农业大学, 2017.
	Chen LC. Cloning and preliminarily functional analysis of SlMYB102 on salt stress in tomato[D]. Tai'an: Shandong Agricultural University, 2017.
[7]	Cui J, Jiang N, Zhou XX, et al. Tomato MYB49 enhances resistance to Phytophthora infestans and tolerance to water deficit and salt stress[J]. Planta, 2018, 248(6): 1487-1503. doi: 10.1007/s00425-018-2987-6
[8]	张旭, 陈丽琛, 任仲海. 番茄过表达SlMYB102对种子萌发及生长的影响[J]. 园艺学报, 2018, 45(8): 1523-1534. doi: 10.16420/j.issn.0513-353x.2018-0001
	Zhang X, Chen LC, Ren ZH. Effects of overexpression of SlMYB102 on the tomato seed germination and growth[J]. Acta Hortic Sin, 2018, 45(8): 1523-1534.
[9]	简伟. 番茄SlMYB75和SlNAC6转录因子在果实成熟及胁迫应答中的功能研究[D]. 重庆: 重庆大学, 2018.
	Jian W. Functional study of tomato SlMYB75 and SlNAC6 transcription factors in fruit ripening and stress responses[D]. Chongqing: Chongqing University, 2018.
[10]	张露月. SlMYB15转录因子调控番茄低温抗性的作用机制研究[D]. 杭州: 浙江大学, 2020.
	Zhang LY. Mechanism of SlMYB15 mediated-regulation of cold response in tomato[D]. Hangzhou: Zhejiang University, 2020.
[11]	刁鹏飞. SlMYB41基因的克隆及其在番茄低温胁迫响应中的功能分析[D]. 泰安: 山东农业大学, 2020.
	Diao PF. Cloning of SlMYB4 and its functional analysis in tomato response to chilling stress[D]. Tai'an: Shandong Agricultural University, 2020.
[12]	Wang ML, Hao J, Chen XH, et al. SlMYB102 expression enhances low-temperature stress resistance in tomato plants[J]. PeerJ, 2020, 8: e10059. doi: 10.7717/peerj.10059 URL
[13]	沈峰屹. 番茄SLMYBl4基因响应非生物胁迫的功能分析[D]. 哈尔滨: 东北农业大学, 2021.
	Shen FY. Functional analysis of SLMYBl4 gene of tomato in response to abiotic stress[D]. Harbin: Northeast Agricultural University, 2021.
[14]	Chen YN, Li L, Tang BY, et al. Silencing of SlMYB55 affects plant flowering and enhances tolerance to drought and salt stress in tomato[J]. Plant Sci, 2022, 316: 111166. doi: 10.1016/j.plantsci.2021.111166 URL
[15]	贾梦玫, 郝娟, 郭仰东, 等. 番茄SlMYB74转录激活分析及互作蛋白的初步筛选[J]. 分子植物育种, 2021. http://kns.cnki.net/kcms/detail/46.1068.S.20211123.1838.014.html.
	Jia MM, Hao J, Guo YD, et al. Analysis of tomato SlMYB74 transcription activation and preliminary screening of interacting proteins[J]. Mol plant breed, 2021. http://kns.cnki.net/kcms/detail/46.1068.S.20211123.1838.014.html.
[16]	张恒, 陈艳琦, 任杰莹, 等. 西南麦区小麦苗期氮高效品种筛选及指标体系构建[J]. 四川农业大学学报, 2022, 40(1): 10-18, 27.
	Zhang H, Chen YQ, Ren JY, et al. Screening of wheat cultivars with high nitrogen efficiency at seedling stage and construction of index system in southwest wheat region[J]. J Sichuan Agric Univ, 2022, 40(1): 10-18, 27.
[17]	徐新娟, 李勇超. 2种植物相对电导率测定方法比较[J]. 江苏农业科学, 2014(7): 311-312.
	Xu XJ, Li YC. Comparison of two relative conductivity measurements in plants. Jiangsu Agric Sci, 2014(7): 311-312.
[18]	苍晶, 赵会杰. 植物生理学实验教程[M]. 北京: 高等教育出版社, 2013.
	Cang J, Zhao HJ. Experimental course of plant physiology[M]. Beijing: Higher Education Press, 2013.
[19]	Kong FY, Deng YS, Zhou B, et al. A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress[J]. J Exp Bot, 2014, 65(1): 143-158. doi: 10.1093/jxb/ert357 pmid: 24227338
[20]	Ahmad A, Hadi F, Ali N. Effective phytoextraction of cadmium(Cd)with increasing concentration of total phenolics and free proline in Cannabis sativa(L)plant under various treatments of fertilizers, plant growth regulators and sodium salt[J]. Int J Phytoremediation, 2015, 17(1/2/3/4/5/6): 56-65. doi: 10.1080/15226514.2013.828018 URL
[21]	罗群. 考马斯亮蓝法快速测定菜籽粕中可溶性蛋白质的含量[J]. 成都大学学报: 自然科学版, 2014, 33(2): 125-126, 129.
	Luo Q. Rapid determination of soluble protein content in rapeseed meal by coomassie brilliant blue method[J]. J Chengdu Univ Nat Sci Ed, 2014, 33(2): 125-126, 129.
[22]	Li CN, Ng CKY, Fan LM. MYB transcription factors, active players in abiotic stress signaling[J]. Environ Exp Bot, 2015, 114: 80-91. doi: 10.1016/j.envexpbot.2014.06.014 URL
[23]	Imahori Y, Takemura M, Bai JH. Chilling-induced oxidative stress and antioxidant responses in mume(Prunus mume)fruit during low temperature storage[J]. Postharvest Biol Technol, 2008, 49(1): 54-60. doi: 10.1016/j.postharvbio.2007.10.017 URL
[24]	赵春梅. 内质网小分子热激蛋白基因导入番茄的研究[D]. 济南: 山东师范大学, 2003.
	Zhao CM. Studies on introduction the gene encoding small heat shock protein in the endoplasmic Reticulum into tomato[D]. Jinan: Shandong Normal University, 2003.
[25]	钱芝龙, 丁犁平, 曹寿椿. 低温胁迫对辣(甜)椒幼苗膜脂过氧化水平及保护酶活性的影响[J]. 园艺学报, 1994, 21(2): 203-204.
	Qian ZL, Ding LP, Cao SC. Effect of low temperature stress on pepper seedling lipid peroxidation level and protective enzyme activity[J]. Acta Hortic Sin, 1994, 21(2): 203-204.
[26]	Alonso A, Queiroz CS, et al. Chilling stress leads to increased cell membrane rigidity in roots of coffee(Coffea arabica L.) seedlings[J]. Biochim Biophys Acta, 1997, 1323(1): 75-84. pmid: 9030214
[27]	王孝宣, 李树德, 东惠茹, 等. 番茄品种耐寒性与ABA和可溶性糖含量的关系[J]. 园艺学报, 1998(1): 56-60.
	Wang XX, Li SD, Dong HR, et al. The correlationship of cold-tolerance with ABA, soluble sugar and respiratory intensity in tomato[J]. Acta Hortic Sin, 1998(1): 56-60.
[28]	Sun Q, Ye ZH, Wang XR, et al. Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii[J]. J Plant Physiol, 2007, 164(11): 1489-1498. doi: 10.1016/j.jplph.2006.10.001 URL
[29]	Gupta NK, Agarwal S, et al. Effect of short-term heat stress on growth, physiology and antioxidative defence system in wheat seedlings[J]. Acta Physiol Plant, 2013, 35(6): 1837-1842. doi: 10.1007/s11738-013-1221-1 URL
[30]	Hasanuzzaman M, Nahar K, Alam M, et al. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants[J]. Int J Mol Sci, 2013, 14(5): 9643-9684. doi: 10.3390/ijms14059643 pmid: 23644891
[31]	Hayat S, Hayat Q, Alyemeni MN, et al. Role of proline under changing environments: a review[J]. Plant Signal Behav, 2012, 7(11): 1456-1466. doi: 10.4161/psb.21949 pmid: 22951402
[32]	钟胜. 抗寒相关基因CBF3、ICE1以及AtGolS3的转基因拟南芥耐寒效果评价[D]. 海口: 海南大学, 2008.
	Zhong S. Evaluation of cold resistance effects of transgenic Arabidopsis with cold resistance-related genes CBF3, ICE1 and AtGolS3[D]. Haikou: Hainan University, 2008.
[33]	Kumar D, Yusuf M, Singh P, et al. Histochemical detection of superoxide and H₂O₂ accumulation in Brassica juncea seedlings[J]. BIO-PROTOCOL, 2014, 4(8): 1108.
[34]	Shulaev V, Oliver DJ. Metabolic and proteomic markers for oxidative stress. New tools for reactive oxygen species research[J]. Plant Physiol, 2006, 141(2): 367-372. pmid: 16760489
[35]	于乔乔. 低温胁迫下玉米幼苗光合及呼吸代谢特性的研究[D]. 哈尔滨: 东北农业大学, 2021.
	Yu QQ. Study on the characteristics of photosynthesis and respiratory metabolism of maize seedlings under low temperature stress[D]. Harbin: Northeast Agricultural University, 2021.
[36]	张娟, 徐坤, 孙杰. 番茄不同砧木材料幼苗对低温胁迫的反应[J]. 西北农业学报, 2004, 13(2): 104-108.
	Zhang J, Xu K, Sun J. Response of different tomato stocks to low temperature stress[J]. Acta Agric Boreali Occidentalis Sin, 2004, 13(2): 104-108.