转录因子<i>SlMYB-related 2</i>对番茄耐冷性的影响

doi:10.7668/hbnxb.20192215

摘要/Abstract

摘要：

番茄属于冷敏感模式植物,在生长过程中极易受冷害进而影响其产量。为进一步揭示番茄植株抗寒的分子机制以及对选育番茄抗寒品种提供理论依据,以蔬菜遗传育种与生物技术实验室种质资源编号25的番茄品种为材料、番茄转录因子SlMYB-related 2为研究对象,基于CRISPR/Cas9基因敲除载体,通过农杆菌转化法转化获得番茄阳性植株;构建VIGS瞬时沉默表达载体,以野生型和未插入目的基因片段的病毒转化植株为对照,将3组植株进行4 ℃(16 h光照/8 h黑暗处理,60%湿度)低温处理后,测定抗冷相关生理指标的含量,比较其耐寒性。结果表明,构建了CRISPR/Cas9基因敲除载体,共获得了CRISPR沉默阳性植株2株;构建了VIGS沉默表达载体,经低温处理后发现,随着低温处理时间的延长,SlMYB-related 2基因转化组番茄植株的脯氨酸和可溶性糖的增长趋势比对照番茄组(野生型携带TRV空载,CK)和野生组(WT)慢,且含量低于CK组和野生组,但丙二醛含量高于CK组和野生组,证明VIGS瞬时表达番茄植株抗寒性显著低于CK组和野生组。通过对基因瞬时沉默表达后番茄植株的抗寒性分析,发现SlMYB-related 2基因在低温胁迫中起到了正调控作用。

关键词: 番茄, MYB转录因子, VIGS, CRISPR/Cas9, 沉默载体, 遗传转化

Abstract:

Tomato belongs to the cold sensitive model plant,which is vulnerable to chilling injury during its growth,thus affecting its yield.In order to provide a theoretical basis for further revealing the molecular mechanism of cold resistance of tomato plants and breeding tomato cold resistant varieties,tomato germplasm resource No.25 of vegetable genetics,Breeding and Biotechnology Laboratory was used as material,and tomato transcription factor SlMYB-related 2 was used as research object.Based on CRISPR/Cas9 gene knockout vector,tomato positive plants were obtained by Agrobacterium transformation.The VIGS transient silencing expression vector was constructed,and the wild-type and and virus transformed plants without inserted target gene fragment were used as control.The three groups of plants were treated with low temperature at 4 ℃(16 h day / 8 h night,60% humidity).The contents of physiological indexes related to cold resistance were measured and its cold tolerance was compared.The results showed that CRISPR/Cas9 gene knockout vector was constructed and two CRISPR silenced positive plants were obtained.Constructed the expression vector of VIGS silence,after low temperature treatment,it was found that with the extension of low temperature treatment time,the growth trend of proline and soluble sugar in SlMYB-related 2 gene transformation group was slower than that in control tomato group(wild type carrying TRV,CK)and wild group(WT),and the content was lower than that in CK group and wild group.However,the content of malondialdehyde was higher than that of CK group and wild group,which proved that the cold resistance of tomato plants with transient expression of VIGS was significantly lower than that of CK group and wild group.Through the analysis of cold resistance of tomato plants after gene transient silencing expression,it was found that SlMYB-related 2 gene played a positive regulatory role in low temperature stress.

Key words: Tomato, MYB transcription factor, VIGS, CRISPR/Cas9, Silence carrier, Genetic transformation

吴红红, 段学粉, 郭仰东, 张喜春. 转录因子SlMYB-related 2对番茄耐冷性的影响[J]. 华北农学报, 2022, 37(1): 35-41. doi: 10.7668/hbnxb.20192215.

WU Honghong, DUAN Xuefen, GUO Yangdong, ZHANG Xichun. Effects of Transcription Factor SlMYB-related 2 on Cold Tolerance of Tomato[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 35-41. doi: 10.7668/hbnxb.20192215.

http://www.hbnxb.net/CN/Y2022/V37/I1/35

图/表 11

图1 CRISPR/Cas9载体示意及sgRNA靶位点信息
LB.T-DNA左边界;Cas9.优化Cas9;RB.T-DNA右边界;NOS Ter.NOS终止子;U6.拟南芥U6启动子;35S.CaMV 35S启动子;SG.向导RNA;Hygro.潮霉素选择标记;e35S.增强子35S启动子;Poly Ter.PolyA终止子。

Fig.1 Diagram of CRISPR/Cas9 vector and information of the sgRNA target site
LB.Left border of T-DNA;Cas9.Optimized Cas9;RB.Right border of T-DNA;NOS Ter.NOS terminator;U6.Arabidopsis U6 promoter;35S.CaMV 35S promoter;SG.sgRNA;Hygro.Hygromycin selection marker;e35S.Enhanced 35S promoter;PolyA Ter.PolyA terminator.

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence
上游靶点Up target	TGATTGCATTGGCATTCCCACCTTGT
下游靶点Low target	AAACACAAGGTGGGAATGCCAATGCA

表2 引物二聚体反应体系

Tab.2 Primer dimer reaction system

成分 Component	体积/μL Volume
退火缓冲液 Buffer anneal	18
上游靶点Up target	1
下游靶点Low target	1

表3 CRISPR双元载体构建

Tab.3 Binary vector construction of CRISPR

成分 Component	体积/μL Volume
CRISPR/Cas载体 CRISPR/Cas vector	2
靶点二聚体Target dimer	1
酶 Enzyme Mix	1
水 ddH₂O	6
总体积 Total volume	10

图2 CRISPR/Cas9基因敲除载体序列比对

Fig.2 CRISPR/Cas9 gene knockout vector sequence alignment

图3 转基因植株的阳性验证
M.Marker;1—2.1号转基因植株;3—4.2号转基因植株。

Fig.3 Validation of positive transgenic plants
M.Marker;1—2.Transgenic plant 1;3—4.Transgenic plant 2.

图4 VIGS瞬时沉默表达载体序列比对

Fig.4 VIGS transient silence expression vector sequence alignment

图5 TRV病毒检测
M.Marker;1.WT;2、3、4、6、7.TRV阳性;5.TRV阴性。

Fig.5 Detection of TRV virus
M.Marker;1.WT;2,3,4,6,7.TRV positive;5.TRV negative.

图6 不同植株材料游离脯氨酸含量比较
不同小写字母表示0.05水平差异显著性。图7—8同。

Fig.6 Comparison of free proline content in different plant materials
Different lowercase letters indicate significant differences in 0.05 levels. The same as Fig.7—8.

图7 不同植株材料丙二醛含量比较

Fig.7 Comparison of malondialdehyde content in different plant materials

图8 不同植株材料可溶性糖含量比较

Fig.8 Comparison of soluble sugar content of different plant materials

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