Effects of Transcription Factor SlMYB-related 2 on Cold Tolerance of Tomato

doi:10.7668/hbnxb.20192215

Abstract

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

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.

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Figures/Tables 11

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.

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

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

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

Fig.2 CRISPR/Cas9 gene knockout vector sequence alignment

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

Fig.4 VIGS transient silence expression vector sequence alignment

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

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.

Fig.7 Comparison of malondialdehyde content in different plant materials

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

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