植物中分布最广泛的一类MYB转录因子是R2R3-MYB蛋白，对植物生长发育的各阶段均起决定性作用，如参与合成次生代谢物质、响应多个逆境胁迫、参与植物的激素应答等[3]。
吴小亲[4]通过对番茄SlMYB64过表达植株的研究发现，其参与番茄叶片及花的发育，为研究基因在花粉发育中的功能奠定了基础。陈静[5]研究结果显示，番茄SlMYB1R-1参与番茄干旱和盐胁迫的响应，在非生物胁迫中发挥负调控作用。陈丽琛[6]研究发现，番茄SlMYB102参与种子萌发的调控，且能提高植株对NaCl胁迫的抗性。2018年Cui等[7]以拟南芥（Arabidopsis thaliana L.）转录因子R2R3-MYBS（AtMYBS）的同缘关系为基础，在番茄基因组中鉴定24个R2R3-MYB转录因子。同年张旭等[8]发现番茄SlMYB102过表达株系中，种子萌发速率明显升高，而植株高度、叶面积、地上部及地下部的鲜量均显著降低。简伟[9]研究证实番茄SlMYB75转录因子能够提高果实花青素含量，改善果实成熟过程中部分品质属性，在采后果实抗病耐贮藏方面作用显著。张露月[10]研究发现低温诱导了番茄SlMYB15的表达，且是低温抗性的一个正向调控因子，HY5-MYB15-CBFs转录级联可增强番茄低温抗性。刁鹏飞[11]通过研究SlMYB41转录因子过表达株系和RNAi转基因株系，发现其不仅是低温诱导表达的负调控因子，同时高温和NaCl也会增强表达。Wang 等[12]研究了番茄SlMYB102过表达可以提高植株抗寒性，同时不会伤害幼苗生长。沈峰屹[13]利用生物信息学分析，鉴定出127个番茄MYB转录因子，对其中几个进行不同胁迫处理发现，低温胁迫下SlMYB7表达量上升；干旱胁迫处理时SlMYB10表达量相对较高；沉默SlMYB14后，植株抗旱性和耐盐性减弱。Chen等[14]发现番茄SlMYB55影响脱落酸（ABA）的生物合成，通过ABA介导的信号转导途径调节干旱和盐类响应，直接或间接影响与干旱和盐类响应、开花时间、萼片大小和花序相关的基因表达，从而调节胁迫耐受性和花序发育。贾梦玫等[15]通过酵母双杂交技术进行番茄SlMYB102互作蛋白的初步筛选，进一步探究SlMYB102参与低温胁迫的调控机制。研究结果显示，MYB转录因子在番茄各个时期参与多种生理反应，响应各种生物胁迫及非生物胁迫，影响番茄整个生长发育过程，为抗逆育种提供了新的技术支持。
本研究利用同源克隆的方法从番茄cDNA中克隆得到SlMYB96，通过荧光定量PCR方法分析该基因的组织表达特异性和不同低温处理时间条件下的表达模式，同时构建该基因的VIGS瞬时沉默体载体，并通过农杆菌介导侵染番茄获得沉默植株，通过对番茄沉默植株抗寒性生理指标的测定，进一步研究SlMYB96的功能，以期为进一步揭示番茄植株抗寒的分子机制以及为选育番茄抗寒品种提供理论依据。
1 材料与方法
1.1 材料
1.1.1 试验材料
所用番茄试验材料为实验室自存俄罗斯品种‘Bolgogragsky’，编号25。
1.1.2 菌株及试剂
大肠杆菌DH5α感受态细胞购自上海昂羽生物技术有限公司，cDNA第一链合成试剂盒购于北京天根生化科技有限公司，无缝克隆试剂盒及各种限制性内切酶购于博迈德公司，TB Green Premix Ex Tap II及Prime STAR Max Premix（2X）购自宝生物（TaKaRa）工程（大连）有限公司。上海生工生物工程股份有限公司（
1.2 方法
1.2.1 材料种植及胁迫处理
在25℃暗培养条件下催生番茄种子3-5 d，发芽后将其播种到穴盘中，于12 000 lx光照强度，25℃光照16 h，20℃黑暗8 h，60%湿度条件下培养。
当番茄幼苗长到四叶一心时，将其置于4℃光照16 h，黑暗8 h，60%湿度条件下进行低温胁迫处理。
1.2.2 番茄总RNA的提取与cDNA第一链的合成
采用传统Trizol试剂在超净工作台（北京德天佑科技发展有限公司JJT-1300型）提取番茄叶片总RNA。使用天根公司试剂盒合成第一链cDNA，反应体系为5×Fast King-RT Super Mix 4 µL、总RNA 1 µL、ddH2O 15 µL。混匀后42℃ 15 min，95℃ 3 min，在PCR仪（美国Bio-Rad公司，C1000 Thermal Cycler）上进行。
1.2.3 SlMYB96的克隆及序列分析
根据番茄基因组数据库（
表1 引物列表
Table 1
| 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 |
注：下划线为限制性内切酶的序列
Note: The sequence of the restriction enzyme is underlined
利用ProtParam在线软件（
1.2.4 SlMYB96的表达模式分析
利用实时荧光定量PCR方法检测SlMYB96在低温胁迫处理0、1、3、5、7、9、12和24 h的表达情况。根据SlMYB96和内参基因Actin序列（表1），设计荧光定量PCR引物（表1），利用TB Green Premix Ex Tap II酶在荧光定量PCR仪（美国Bio-Rad公司，Bio-Rad CFX 96 T）上操作，反应体系为cDNA 2 µL、上游引物1 µL、下游引物1 µL、TB Green Premix Ex Tap II 9 µL和ddH2O 7 µL。反应程序为95℃ 30 s；95℃ 5 s，60℃ 30 s，39个循环；95℃ 10 s，65℃ 0.05 s，95℃ 0.5 s。每个样本进行3个技术重复。
1.2.5 SlMYB96的VIGS载体构建
利用Sol Genomics Network在线软件设计SlMYB96 VIGS特异性表达引物（表1），采用PCR方法对目的片段扩增，扩增产物进行1%琼脂糖凝胶电泳回收，回收方法按照试剂盒进行，将纯化回收的目的基因片段转化大肠杆菌DH5α，将菌液送去测序，将测序正确的菌液提取质粒。对测序正确质粒和TRV2质粒用Xba I和Sma I进行双酶切，用T4连接酶连接、转化，并进行菌落PCR鉴定及测序，将测序正确的菌液提取质粒，-20℃保存，用于后续试验。回收、鉴定、测序方法同1.2.3。
1.2.6 农杆菌介导的VIGS侵染番茄及阳性植株鉴定
将含有pTRV1、pTRV2、pTRV2-MYB96质粒的GV3101农杆菌菌液培养至OD600=0.6-1.0。4 000 r/min离心8 min收集菌体，配制pH为5.6的侵染液（含10 mmol/L MES, 10 mmol/L MgCl2, 200 μmol/L AS），重悬菌体，使得OD600调整为1.0。等体积混合pTRV1和pTRV2、pTRV2-MYB96侵染液，黑暗静置4 h。用注射器注射3-4片真叶番茄。侵染后，于20℃，光照16 h，黑暗8 h，60%相对湿度条件下培养。侵染株数分别为野生型组（WT）50株，空载组（CK）50株，SlMYB96沉默组50株。
侵染10 d后，取野生型组（WT）、空载组（CK）、SlMYB96沉默组3组植株叶片提取RNA，每个样本取3个技术重复，测定浓度后选合适RNA进行反转录cDNA，用PCR方法验证阳性植株。
1.2.7 低温胁迫下番茄植株的生理生化指标测定
将野生型组（WT）、空载组（CK）、SlMYB96瞬时沉默组番茄植株置于培养箱4℃，光照16 h，黑暗8 h，60%湿度的培养条件下培养。低温条件下培养5 d后，上午8：00取样，进行各项生理生化指标测定。其中，叶绿素含量测定参照分光光度法[16]，相对电导率采用电导法[17]测定，丙二醛（malondialdehyde, MDA）和可溶性糖含量参照《植物生理学实验教程》[18]和Kong等[19]测定，脯氨酸含量的测定采用磺基水杨酸[20]的方法，可溶性蛋白含量采用考马斯亮蓝法[21]测定，超氧化物歧化酶（superoxide dismutase, SOD）、过氧化物酶（peroxidase, POD）、过氧化氢酶（catalase, CAT）活性依据试剂盒方法测定。
2 结果
2.1 SlMYB96的克隆及序列分析
以番茄cDNA为模板进行扩增（图1），获得一条清晰条带，且与目的片段大小一致，确定为SlMYB96的CDS序列。
图1
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
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
2.2 SlMYB96的表达模式分析
运用荧光定量PCR技术分析番茄6个不同组织部位SlMYB96的表达水平（图3-A），与营养器官相比，SlMYB96在生殖器官表达量较高。在营养器官叶中的相对表达水平最高，在生殖器官花中的相对表达水平最高，SlMYB96是组织差异性表达基因。
图3
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
此外，经4℃低温处理的根茎叶中SlMYB96受诱导表达，其中，根的诱导表达更为明显，3 h达到最大值，提高16倍。茎在5 h表达量开始受抑制，而叶则在4 h受抑制（图3-B）。说明SlMYB96与番茄抗寒机制有直接关系。
2.3 番茄VIGS载体的构建
图4
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
2.4 农杆菌介导的VIGS基因沉默阳性植株验证
将（pTRV1、pTRV2、pTRV2-MYB96）质粒转化农杆菌并以野生型/空载组/试验组侵染4-5叶一心的番茄叶片，10 d后取样，运用RT-PCR检测阳性植株，结果（图5）表明，野生型（WT）30株，携带pTRV1+pTRV2空载（CK）39株，携带pTRV1+pTRV2-MYB96试验组33株。
图5
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
2.5 低温胁迫下番茄植株的表型变化和生理生化指标测定
2.5.1 低温胁迫下番茄植株的表型变化
4℃低温处理前，野生型株（WT）、空载株（CK）以及基因瞬时沉默（pTRV2-MYB96）3组不同类型植株的茎秆直立挺拔，叶片伸展且色泽鲜艳，生长状况良好（图6）。经4℃低温处理5 d后，野生型组（WT）番茄植株茎秆略有弯曲，叶片颜色变浅，幼苗整体没有明显损伤；空载组（CK）番茄植株茎秆略微倾斜，幼苗叶片卷曲呈褐色且带有少量冻斑，同时，叶片边缘出现黑色冻伤现象；基因瞬时沉默组（pTRV2-MYB96）植株茎秆倾斜，叶柄严重下垂，叶片出现萎蔫并呈现水浸状。从表型结果观察可知，SlMYB96沉默后，番茄植株对低温的耐受性降低。
图6
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
2.5.2 低温胁迫下番茄植株的生理生化指标测定
25℃时，空载组（CK）叶绿素含量最高，其次是野生型组（WT），基因瞬时沉默组（pTRV2-MYB96）叶绿素含量最低（图7-A）。经4℃低温处理5 d后，3组植株的叶绿素含量均发生下降，但基因瞬时沉默组（pTRV2-MYB96）植株的叶绿素含量整体相比野生型组（WT）、空载组（CK）植株而言含量较高。说明植株遭受低温胁迫时，叶片受到不同程度的损伤。
图7
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
25℃时，3组植株中基因瞬时沉默组的叶片相对电导率最高，其次是野生型组（WT），空载组（CK）最低（图7-B）。经低温处理5 d后，3组植株的叶片相对电导率均明显增加，其中空载组（CK）最高，基因瞬时沉默组居于第二。表明空载组（CK）、基因瞬时沉默组植株的膜功能受损较为严重。
丙二醛作为细胞膜的另一个重要的指标，当植物受到伤害时，会出现膜脂过氧化现象。植物低温处理前，3组植株中基因瞬时沉默组（pTRV2-MYB96）的丙二醛含量最高，空载（CK）最低。低温处理后，空载组（CK）丙二醛含量升高，其他2组含量均降低，且基因瞬时沉默组（pTRV2-MYB96）丙二醛含量降低不明显（图7-C）。
在25℃条件下，野生型（WT）植株的脯氨酸含量最高，其次是基因瞬时沉默组，空载（CK）植株最低。但4℃低温处理5 d后，每组植株的脯氨酸的含量明显增加，尤其是在基因瞬时沉默组植株中最为明显，同时低温处理后，野生型组（WT）植株的脯氨酸含量反而最低，基因瞬时沉默组最高，与低温前各组植株脯氨酸含量呈相反结果（图7-D）。
低温处理前3组植株中，基因瞬时沉默植株的可溶性糖含量最高，空载（CK）最低。当受到低温胁迫时，3组植株的可溶性糖含量明显增加，其中，空载（CK）组变化最高，几乎成倍增长；野生型（WT）也较低温处理前有所增长；而基因瞬时沉默组（pTRV2-MYB96）植株的可溶性糖含量因为发生基因沉默并未出现明显增强，但也比低温处理前更高（图7-E）。可能是因为基因瞬时沉默组（pTRV2-MYB96）植株中SlMYB96的沉默而使植株体内自身无法合成更多的可溶性糖抵御寒冷，致使植株受冷害严重。说明SlMYB96沉默后，植株不耐低温。
低温处理前，3组植株的可溶性蛋白含量中野生型（WT）最高，其次是基因瞬时沉默组（pTRV2-MYB96），空载（CK）组最低（图7-F）。但是经低温胁迫处理5 d后，3组植株叶片的可溶性蛋白含量均出现了下降的趋势，其中，空载（CK）组含量降低不明显，但整体来讲，低温处理后的3组植株中可溶性蛋白含量最高的依然是野生型（WT），基因瞬时沉默组（pTRV2-MYB96）含量最低，可能是因为植株受到低温伤害时，蛋白合成受到抑制；抑或是因为SlMYB96发生沉默，植株体内无法合成更多的可溶性蛋白，因而耐冷性相比而言更低。
低温处理前3组植株中野生型（WT）组的过氧化氢酶（CAT）活性最高，其次是空载（CK）组，基因瞬时沉默组（pTRV2-MYB96）植株的活性最低。4℃低温处理5 d后，3组植株的过氧化氢酶（CAT）活性均显著升高，其中野生型组（WT）的活性最高，其次为基因瞬时沉默组（pTRV2-MYB96），空载组（CK）的活性最低（图7-G）。
4℃低温处理前，3组植株中空载组（CK）的超氧化物歧化酶（SOD）活性最高，其次是野生型组（WT），基因瞬时沉默组（pTRV2-MYB96）活性最低。4℃低温处理5 d后，3组植株的超氧化物歧化酶（SOD）活性显著下降，其中基因瞬时沉默组（pTRV2-MYB96）植株活性最大，其次为野生型组（WT），空载组（CK）最低（图7-H）。总之，基因瞬时沉默组（pTRV2-MYB96）植株的下降幅度比其他2组较小。
3组植株在4℃低温处理前，空载组（CK）的过氧化物酶（POD）活性最高，其次是基因瞬时沉默组（pTRV2-MYB96），野生型组（WT）活性最低。4℃低温处理后，野生型组（WT）和基因瞬时沉默组（pTRV2-MYB96）植株的过氧化物酶（POD）活性呈现上升趋势，原因可能是外界低温刺激促进了过氧化物酶（POD）的合成（图7-I）。
3 讨论
预防和抵御植株冷害，需要了解植物基因组水平上对非生物胁迫的反应，目前已有诸多关于基因参与植物冷胁迫的研究，但很多相关抗寒机制仍在摸索当中。已有相关研究表明，MYB蛋白直接或间接参与调节响应非生物胁迫，除此之外，也发现MYB蛋白调控并作用于其下游，具体包括Pi转运蛋白、脱落酸合成蛋白、活性氧清除蛋白、细胞扩增及其他应激诱导蛋白[22]。
膜脂过氧化作用属于一系列自由基反应，其发生在不饱和脂肪酸中，它会氧化细胞膜上的结构骨架磷脂分子，使细胞膜受到破坏，同时只有在特定条件下这种现象才会发生。作为膜脂过氧化最终分解产物之一的丙二醛，当细胞膜透性增加时丙二醛的含量也会随之升高，而且活性氧毒害作用的表现便是丙二醛含量的积累[26]。丙二醛是判定植株在逆境胁迫下脂质过氧化强度的一个重要生理指标，同时也是植株衰老的表现[27]。由于低温胁迫增加了水解酶的活性，促进了淀粉的加速分解，改变了可溶性糖的含量。而当积累一定程度的可溶性糖时，细胞液浓度便会升高，进而使植物的耐寒性提高。在低温条件下，评估番茄耐寒性的另一指标为可溶性糖浓度[28]。渗透调节物质包括可溶性糖和游离脯氨酸，脯氨酸是一种在植物受到非生物胁迫时合成的重要渗透调节物质[29]。积累一定量的脯氨酸对细胞质水势降低、平衡水分、螯合单线态氧、羟自由基的清除、保护酶和蛋白质结构的稳定等具有一定的作用[30⇓-32]。植物膜系统的损伤或膜结构的破坏会使膜透性改变，使细胞内小分子电解质外渗，增大植物细胞浸提液的电导率。膜透性与植物抗逆性强弱有关，抗逆性越强细胞的膜越稳定，膜透性也与逆境胁迫强度有关，胁迫越强膜损伤越严重，膜透性越大。细胞浸出液相对电导率的变化直接反映质膜受伤害的程度，细胞内含物外渗，导致电导率增大，使得植物受伤，抗性越弱[33]。O2-和H2O2作为活性氧的两种主要表现形式，在受到低温胁迫时会过度累积。NBT染色法可以被用作评估O2-的原位积累量的一种有效手段，当染色面积越大时，O2-的积累量就会越多，那么植株受到伤害的程度越严重[34-35]。丙二醛、可溶性糖、游离脯氨酸、叶片活性氧以及相对电导率含量的测定，都可以作为植株抗寒性的鉴定。除了这些生理指标之外，可溶性蛋白和叶绿素的含量也可作为辅助手段帮助进一步鉴定植株的耐寒性。
植物某一性状的变化趋势可以通过单一的生理生化指标反映，但对于植物响应逆境胁迫的综合能力难以反映出来，而同时测定多项生理生化指标，各项指标相互佐证，可以将植物的抗逆性实质更好地反映出来，真正体现植物的特性[36]。本研究通过TRV介导的SlMYB96沉默，观察外观性状并测定野生型组（WT）、空载组（CK）以及基因瞬时沉默组（pTRV2-MYB96）3组植株游离脯氨酸、可溶性糖、相对电导率等各项生理生化指标分析得出，TRV病毒侵染番茄幼苗并低温处理5 d后，基因瞬时沉默组耐寒性较差即SlMYB96属于抗寒基因。
4 结论
从番茄中克隆获得SlMYB96，其CDS全长为1 008 bp，编码335个氨基酸。SlMYB96对低温胁迫有响应，将其沉默之后，会使番茄植株抗寒性降低。
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