生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 236-245.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1051
收稿日期:
2022-08-23
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
张喜春,男,博士,教授,硕士生导师,研究方向:蔬菜遗传育种与生物技术;E-mail: xichunzhang@sina.com作者简介:
胡明月,女,硕士,研究方向:蔬菜遗传育种;E-mail: mingyuehu96@163.com
基金资助:
HU Ming-yue1(), YANG Yu1, GUO Yang-dong2, ZHANG Xi-chun1()
Received:
2022-08-23
Published:
2023-04-26
Online:
2023-05-16
摘要:
研究SlMYB96在抗寒中的作用,为研究番茄的抗寒分子机制及选育番茄抗寒品种提供理论依据。以番茄cDNA为模板克隆SlMYB96,利用生物信息软件分析该基因的理化性质,利用实时荧光定量(RT-qPCR)技和病毒诱导的基因沉默(virus induced gene silencing, VIGS)技术研究低温胁迫处理后SlMYB96的表达特征及其在番茄抗寒过程中的作用。结果表明,SlMYB96在番茄的根、茎、叶、花和果实中均有表达,且在花中表达水平最高;随着4℃低温处理时间的增加,SlMYB96的表达量升高,其中在低温处理3 h时表达量达到最大。借助TRV病毒介导的基因沉默技术将SlMYB96沉默,对野生型组(WT)、空载组(CK)以及基因瞬时沉默组(pTRV-MYB96)3组不同类型番茄植株进行低温胁迫后,外观性状结果显示,在4℃低温处理5 d后,与野生型组(WT)、空载组(CK)相比,基因瞬时沉默组(pTRV-MYB96)植株表现出更为明显的冷害症状;生理水平鉴定结果表明,4℃低温处理番茄幼苗5 d时,基因瞬时沉默组(pTRV-MYB96)植株叶绿素、丙二醛、可溶性蛋白含量和超氧化物歧化酶活性明显变低,而可溶性糖、相对电导率、游离脯氨酸含量以及过氧化氢酶、过氧化物酶活性增加,说明基因瞬时沉默组(pTRV-MYB96)植株与野生型组(WT)、空载组(CK)相比抗寒性较低。证明SlMYB96能够响应低温胁迫,将其沉默后番茄植株抗寒性降低。
胡明月, 杨宇, 郭仰东, 张喜春. 低温胁迫下番茄SlMYB96的功能分析[J]. 生物技术通报, 2023, 39(4): 236-245.
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.
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 |
表1 引物列表
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 |
图2 SlMYB96的序列分析 A:SlMYB96亲水性分析;B:SlMYB96保守结构域分析;C:SlMYB96二级结构;D:SlMYB96三级结构
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
图3 SlMYB96的表达模式分析 A:不同组织中SlMYB96的表达模式分析;B:低温胁迫下SlMYB96的根茎叶表达模式分析。不同小写字母表示在P=0.05水平差异显著,下同
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
图4 pTRV2-MYB96载体的构建 A:pTRV2-MYB96载体的酶切验证;B:pTRV2-MYB96载体序列比对;M:BM2000 DNA marker;1-3:3个重复
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
图5 TRV病毒检测 M:BM2000 DNA marker;1-2:WT;3-6、8、9:TRV阳性;7、10:TRV阴性
Fig. 5 TRV virus detection M: BM2000 DNA marker. 1-2: WT. 3-6, 8, 9: TRV positive. 7, 10: TRV negative
图6 VIGS沉默番茄植株表型观察 A-C:25℃野生组、空载组、瞬时沉默植株;D-F:4℃处理5 d后野生组、空载组、瞬时沉默植株
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
图7 低温胁迫下番茄植株的生理生化指标测定 A:叶片叶绿素含量;B:叶片相对电导率;C:叶片丙二醛含量;D:叶片游离脯氨酸含量;E:叶片可溶性糖含量;F:叶片可溶性蛋白含量;G:叶片过氧化氢酶活性;H:叶片超氧化物酶活性;I:叶片过氧化物酶活性
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
[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. |
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