Function Analysis of SlWRKY46 in Regulating Tomato Response to Low Temperature Stress

doi:10.16420/j.issn.0513-353x.2024-0014

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (12): 2758-2774.doi: 10.16420/j.issn.0513-353x.2024-0014

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Function Analysis of SlWRKY46 in Regulating Tomato Response to Low Temperature Stress

SUN Huiru¹^,²(), DANG Fengfeng¹^,², REN Min¹^,², ZHANG Jianing¹, FAN Bei¹, CHEN Guoliang¹^,², CHENG Guoting¹^,², WANG Yanfeng¹^,²()

¹College of Life Sciences，Yan’an University，Yan’an，Shaanxi 716000，China
²Shaanxi Key Laboratory of Research and Utilization of Resource Plants on the Loess Plateau，College of Life Sciences，Yan’an University，Yan’an，Shaanxi 716000，China

Received:2024-03-04 Revised:2024-09-23 Online:2024-12-25 Published:2024-12-13
Contact: SUN Huiru, WANG Yanfeng

Abstract

Abstract:

In order to explore the function of SlWRKY46 in response to low temperature stress，the CDS sequence of SlWRKY46 cloned from tomato‘Micro-Tom’leaves was analyzed. The result showed that SlWRKY46 belonged to Ⅱa group of WRKY family and encoded 253 amino acids. SlWRKY46 was localized in the nucleus by subcellular localization analysis. SlWRKY46 had no transcriptional activation activity in yeast. SlWRKY46 could form homologous dimers by yeast two-hybrid yeast（Y2H）. The results of real-time fluorescence quantitative PCR（qRT-PCR）showed that the expressions of SlWRKY46 were significantly induced by low temperature and abscisic acid（ABA）. The GUS staining of SlWRKY46 promoter transgenic tomato also cloud be induced by low temperature and ABA. The overexpressed SlWRKY46 tomato lines had higher cold tolerance compared with wild-type. The overexpressed SlWRKY46 tomato lines exhibited lower reactive oxygen species accumulation and malondialdehyde levels，and higher antioxidant enzyme activities and proline accumulation compared with wild-type under low temperature treatment. The expression levels of genes related to antioxidant enzyme and ABA showed significant differences between overexpressed SlWRKY46 tomato lines and wild-type under low temperature treatment. Taken together，these results indicated that SlWRKY46 positively regulated the cold tolerance of tomato by antioxidant enzyme and ABA pathways.

Key words: tomato, SlWRKY46, low temperature, reactive oxygen species, ABA

SUN Huiru, DANG Fengfeng, REN Min, ZHANG Jianing, FAN Bei, CHEN Guoliang, CHENG Guoting, WANG Yanfeng. Function Analysis of SlWRKY46 in Regulating Tomato Response to Low Temperature Stress[J]. Acta Horticulturae Sinica, 2024, 51(12): 2758-2774.

Figures/Tables 10

References 57

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用途 Application	基因名称/ID Gene name/ID	引物序列（5′-3′） Primer sequence
CDS扩增 CDS amplification	SlWRKY46	F：ATGGGAAACAAGTCTTTTTTTA；R：TCAAAAAAGTAATCCTGAGATG
酵母载体构建 Yeast vector construct	SlWRKY46-BD	F：CATGGAGGCCGAATTCATGGGAAACAAGTCTTTTTTTA； R：GCAGGTCGACGGATCCTCAAAAAAGTAATCCTGAGATG
亚细胞定位载体构建Subcellular localization vector construction	SlWRKY46-subcell	F：CAGTGGTCTCACAACATGGGAAACAAGTCTTTTTTTATTGACCTTAAC ACAAATCC； R：CAGTGGTCTCATACAAAAAAGTAATCCTGAGATGGCTGTAGCAACTG
过表达载体构建 Overexpressing vector construction	SlWRKY46-pBI121	F：CACGGGGGACTCTAGAATGGGAAACAAGTCTTTTTTTA； R：GACGGCCAGTGAGCTCTCAAAAAAGTAATCCTGAGATG
启动子扩增 Promoter amplication	SlWRKY46-promoter	F：CAAATAGTTAGCTAGTAGCT；R：ACTTGTGTCTATGGAATATA
启动子载体构建 Promoter vector construction	SlWRKY46-promoter-pBI121	F：TGATTACGCCAAGCTTCAAATAGTTAGCTAGTAGCT； R：GGACTGACCACCCGGGACTTGTGTCTATGGAATATA
内参基因 Reference gene	SlEF1α-RT	F：GACAGGCGTTCAGGTAAGG；R：CCAATGGAGGGTATTCAGC
荧光定量PCR qRT-PCR	SlWRKY46-RT	F：ACAATCATCCCCAACCACCT；R：AAGACGCCATTTGTTCGACT
	SlPYL/Solyc06g050500	F：CGGTGGTGGTAGAGTCGTATG；R：AGCCAAGTTCTGTGCGATTTG
	SlPP2C/Solyc01g065700	F：TGCTTCTCACAACTTGGCTGT；R：AAGGACCATGCCCATCAAACA
	SlPP2C/Solyc05g018300	F：TCATCGAAGTCCTGTTCCTGC；R：CCTCCATGTCACGATTAGGGG
	SlPP2C/Solyc06g051940	F：ACTTGCCAGTGATGGTCTGTG；R：TCTGTACTGCCCCGTGAAATC
	SlNCEB/Solyc01g087260	F：GGAGTGGCGAGAATTGAAGGA；R：GGAGTTTCATCGGTAGGTGCA
	SlNCEB/Solyc08g075490	F：AGACGAACTTCCTCCTACCGA；R：AGCATTCCATCACCGTCGAAT
	SlPOD/Solyc02g079500	F：AACACATGCCCCAACGTATCT；R：AGCACCACTAAAGTTGGCAGT
	SlPOD/Solyc02g083490	F：AACATGCCCTCAAGCTGAAGA；R：AGAAGCATCACAACCCCGTAG
	SlPOD/Solyc05g046020	F：GCCAAGAGTTGTCCAAAAGCA；R：TGCATCACATCCCCTGACAAA
	SlPOD/Solyc07g052510	F：GGAGAGGTCTGTTCCAATCCG；R：GCTTCCTGATTTCACCAGCAC
	SlPOD/Solyc10g084200	F：GCCTAGTATAACGGTGCCACA；R：CCTCGACAGCCTATCACCTTG
	SlSOD/Solyc11g066390	F：TGACCTGGGAAACATAGTGGC；R：CAAGTGCTCGTCCAACAACTG
	SlCAT/Solyc02g082760	F：ACAGGGATGAGGAGATCGACT；R：TGTCTGTCGGGTGTGAATGAG
	SlICE1/Solyc03g118310	F：CAGGCTTTACATGCTGCGAT；R：GGTGTCAGAGGATGGAAGCT
	SlICE1/Solyc06g068870	F：GGAGCACAACCAACCCTTTT；R：TTCCCCACTACCCCACTTTC
	SlDREB/Solyc12g008350	F：AAAAGCCGCTCACATGGATC；R：GAGGATGTAACGCCCGAAAC
	SlDREB/Solyc12g044390	F：TGCGAAGTTGGGGAAAATGG；R：AGGTGAAATTGACGCTGGAC
	SlDREB/Solyc03g120840	F：CCCACCTTCTTCATCCTCGT；R：CCTTCAACCGATTCGCTCAG
	SlDREB/Solyc06g066540	F：ATGGGTGTCTGAAATTCGCG；R：GCAGCTTGAACATCTCTCGG
	SlDREB/Solyc08g066660	F：GCAGCAATGGACAAGTTCGA；R：TGTTGCCAAATCTATCGCCG
	SlCOR47-like/Solyc04g082200	F：TCTAGTAGCTCCAGTGATG；R：TCTCCTCTGTTTCCTCGT
	SlCOR413-like/Solyc02g071210	F：AGCAGCTTATCTGGGTTT；R：AATGATGGCTAGGGTGGC
	SlCBF1/Solyc03g026280	F：GAGTCGGAAGAAGTTTCAGG；R：TGTAGGCATCAGTTTCCAC
	SlCBF2/Solyc03g124110	F：TTTCCTACGGCTGAAATGGC；R：GAGTTGGAGGAAGCAGGGAT
	SlCBF3/Solyc03g026270	F：TGCCGGGTTTACTTACGAAT；R：TCAGCTTCCACATGATCTCC

用途 Application	基因名称/ID Gene name/ID	引物序列（5′-3′） Primer sequence
CDS扩增 CDS amplification	SlWRKY46	F：ATGGGAAACAAGTCTTTTTTTA；R：TCAAAAAAGTAATCCTGAGATG
酵母载体构建 Yeast vector construct	SlWRKY46-BD	F：CATGGAGGCCGAATTCATGGGAAACAAGTCTTTTTTTA； R：GCAGGTCGACGGATCCTCAAAAAAGTAATCCTGAGATG
亚细胞定位载体构建Subcellular localization vector construction	SlWRKY46-subcell	F：CAGTGGTCTCACAACATGGGAAACAAGTCTTTTTTTATTGACCTTAAC ACAAATCC； R：CAGTGGTCTCATACAAAAAAGTAATCCTGAGATGGCTGTAGCAACTG
过表达载体构建 Overexpressing vector construction	SlWRKY46-pBI121	F：CACGGGGGACTCTAGAATGGGAAACAAGTCTTTTTTTA； R：GACGGCCAGTGAGCTCTCAAAAAAGTAATCCTGAGATG
启动子扩增 Promoter amplication	SlWRKY46-promoter	F：CAAATAGTTAGCTAGTAGCT；R：ACTTGTGTCTATGGAATATA
启动子载体构建 Promoter vector construction	SlWRKY46-promoter-pBI121	F：TGATTACGCCAAGCTTCAAATAGTTAGCTAGTAGCT； R：GGACTGACCACCCGGGACTTGTGTCTATGGAATATA
内参基因 Reference gene	SlEF1α-RT	F：GACAGGCGTTCAGGTAAGG；R：CCAATGGAGGGTATTCAGC
荧光定量PCR qRT-PCR	SlWRKY46-RT	F：ACAATCATCCCCAACCACCT；R：AAGACGCCATTTGTTCGACT
	SlPYL/Solyc06g050500	F：CGGTGGTGGTAGAGTCGTATG；R：AGCCAAGTTCTGTGCGATTTG
	SlPP2C/Solyc01g065700	F：TGCTTCTCACAACTTGGCTGT；R：AAGGACCATGCCCATCAAACA
	SlPP2C/Solyc05g018300	F：TCATCGAAGTCCTGTTCCTGC；R：CCTCCATGTCACGATTAGGGG
	SlPP2C/Solyc06g051940	F：ACTTGCCAGTGATGGTCTGTG；R：TCTGTACTGCCCCGTGAAATC
	SlNCEB/Solyc01g087260	F：GGAGTGGCGAGAATTGAAGGA；R：GGAGTTTCATCGGTAGGTGCA
	SlNCEB/Solyc08g075490	F：AGACGAACTTCCTCCTACCGA；R：AGCATTCCATCACCGTCGAAT
	SlPOD/Solyc02g079500	F：AACACATGCCCCAACGTATCT；R：AGCACCACTAAAGTTGGCAGT
	SlPOD/Solyc02g083490	F：AACATGCCCTCAAGCTGAAGA；R：AGAAGCATCACAACCCCGTAG
	SlPOD/Solyc05g046020	F：GCCAAGAGTTGTCCAAAAGCA；R：TGCATCACATCCCCTGACAAA
	SlPOD/Solyc07g052510	F：GGAGAGGTCTGTTCCAATCCG；R：GCTTCCTGATTTCACCAGCAC
	SlPOD/Solyc10g084200	F：GCCTAGTATAACGGTGCCACA；R：CCTCGACAGCCTATCACCTTG
	SlSOD/Solyc11g066390	F：TGACCTGGGAAACATAGTGGC；R：CAAGTGCTCGTCCAACAACTG
	SlCAT/Solyc02g082760	F：ACAGGGATGAGGAGATCGACT；R：TGTCTGTCGGGTGTGAATGAG
	SlICE1/Solyc03g118310	F：CAGGCTTTACATGCTGCGAT；R：GGTGTCAGAGGATGGAAGCT
	SlICE1/Solyc06g068870	F：GGAGCACAACCAACCCTTTT；R：TTCCCCACTACCCCACTTTC
	SlDREB/Solyc12g008350	F：AAAAGCCGCTCACATGGATC；R：GAGGATGTAACGCCCGAAAC
	SlDREB/Solyc12g044390	F：TGCGAAGTTGGGGAAAATGG；R：AGGTGAAATTGACGCTGGAC
	SlDREB/Solyc03g120840	F：CCCACCTTCTTCATCCTCGT；R：CCTTCAACCGATTCGCTCAG
	SlDREB/Solyc06g066540	F：ATGGGTGTCTGAAATTCGCG；R：GCAGCTTGAACATCTCTCGG
	SlDREB/Solyc08g066660	F：GCAGCAATGGACAAGTTCGA；R：TGTTGCCAAATCTATCGCCG
	SlCOR47-like/Solyc04g082200	F：TCTAGTAGCTCCAGTGATG；R：TCTCCTCTGTTTCCTCGT
	SlCOR413-like/Solyc02g071210	F：AGCAGCTTATCTGGGTTT；R：AATGATGGCTAGGGTGGC
	SlCBF1/Solyc03g026280	F：GAGTCGGAAGAAGTTTCAGG；R：TGTAGGCATCAGTTTCCAC
	SlCBF2/Solyc03g124110	F：TTTCCTACGGCTGAAATGGC；R：GAGTTGGAGGAAGCAGGGAT
	SlCBF3/Solyc03g026270	F：TGCCGGGTTTACTTACGAAT；R：TCAGCTTCCACATGATCTCC

Function Analysis of SlWRKY46 in Regulating Tomato Response to Low Temperature Stress

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