低温胁迫下番茄SlMYB96的功能分析

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

摘要/Abstract

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。

关键词: 番茄, SlMYB96, 功能分析, 抗寒性

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

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.

图/表 8

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1 SlMYB96的扩增 M：BM2000 DNA marker；1-3：3个重复

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

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

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