Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4265-4273.doi: 10.3864/j.issn.0578-1752.2021.20.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

IbMKP6, A Mitogen-Activated Protein Kinase, Confers Low Temperature Tolerance in Sweetpotato

JIN Rong(),LIU Ming,ZHAO Peng,ZHANG QiangQiang,ZHANG AiJun,TANG ZhongHou()   

  1. Xuzhou Institute of Agricultural Sciences of Xuhuai District of Jiangsu Province/Xuzhou Sweetpotato Research Center of Jiangsu Province/Key Laboratory of Sweetpotato Biology and Genetic Breeding, Ministry of Agriculture, Xuzhou 221131, Jiangsu
  • Received:2021-04-06 Accepted:2021-06-21 Online:2021-10-16 Published:2021-10-25
  • Contact: ZhongHou TANG E-mail:jinrong_2012@126.com;zhonghoutang@sina.com

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Abstract:

【Objective】Studying the function of mitogen activated protein kinase (MAPK) IbMPK6 in respect of low temperature stress tolerance in sweetpotato, that will help us to understand the mechanism of adaption to low temperature stress and play a fundamental role in molecular breeding of sweetpotato. 【Method】Agrobacterium tumefaciens strain EHA105 harbored the plasmid 35S::IbMPK6-GFP were transformed intosweetpotato cv. Xushu29 embryogenic callus. Molecular examination and qRT-PCR were used to screen and select transgenic lines. For low temperature stress assay, selected transgenic lines were performed to observe the phenotype and determine the physiological indexes such as Fv/Fm, the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) after low temperature treatment and recovery treatment. Diaminobenzidine (DAB) staining and nitro blue tetrazolium (NBT) staining analysis were performed to observe reactive oxygen species (ROS) accumulation. The expression level of the key transcription factor IbCBF3 and downstream gene IbCOR27 involved in low temperature signal transduction pathway were identified before and after low temperature treatment. 【Result】Twelve transgenic lines were generated and three transgenic lines (L3, L8 and L11) with a high expression level of IbMPK6 were selected for low temperature tolerance assay. Under low temperature stress, the level of Fv/Fm in transgenic lines L3, L8 and L11 was 0.79, 0.79 and 0.80, while that in WT was 0.05. After temperature recovery treatment, Fv/Fm in transgenic lines has recovered to former levels, whereas the level of Fv/Fm in WT was only 0.70, which was significantly lower than that in transgenic lines. MDA content of three transgenic (lines L3, L8 and L11) increased by 0.02, 0.04 and 0.02 μmol·g-1, and it of WT increased by 0.05 μmol·g-1 after low temperature stress treatment, respectively. After recovery treatment, MDA content in transgenic lines was 0.01 μmol·g-1 on average, whereas it of WT was 0.03 μmol·g-1. The results of DAB and NBT staining showed that the leaves of WT were stained deeper than those of transgenic lines, indicated that hydrogen peroxide and superoxide anion were accumulation less in transgenic lines than in WT. Furthermore, H2O2 level in WT was significantly higher than that in transgenic lines under low temperature stress condition and after recovery treatment. Low temperature regulated the expression level of IbCBF3 and IbCOR27 genes, but the expression level in transgenic lines was higher than that in WT. 【Conclusion】Overexpression of IbMPK6 in sweetpotato resulted in enhanced tolerance to low temperature stress, via alleviating the damage of membrane and photosynthetic system, and decreasing ROS accumulation. IbMPK6 involved in low temperature signaling transduction pathway by up-regulating the expression level of cold related genes IbCBF3 and IbCOR27.

Key words: sweetpotato, IbMPK6, transgenic lines, low temperature stress

Table 1

The primer sequences"

引物名称 Gene name 正向引物序列 Forward primer sequence (5′-3′) 反向引物序列 Revers primer sequence (5′-3′)
IbMPK6-PCR ATGGACGCTGGTTCGGCTCAG TCACATTTGCAGCTCAAACTC
GFP6-PCR AGGTTATTGGAAAATTAAGGGCC AACCAGATCCGATTTTGGAGGATG
IbMPK6-RT GCGGCAGATTCATCCAATAC TATCGCTACGTGCTCATTCG
IbCBF3 TTCGCCACTGTCTTCTTC TATCCTGGACTTCTTGTTG
IbCOR27 CCAATCTCAGCTTCCTTTGC CATGAACCTCAGCATTGTCG
IbARF CTTTGCCAAGAAGGAGATGC TCTTGTCCTGACCACCAACA

Fig. 1

Generation of IbMPK6-overexpression transgenic sweetpotato plants A: Schematic diagram of IbMPK6-overexprssing constructs. B: Genomic DNA PCR analysis of IbMPK6-overexpressing transgenic sweetpotato lines. M: Marker; P: Plasmid; WT: Wide type; L1-L12: Transgenic plants. C: qRT-PCR analysis of IbMPK6-overexpressing transgenic sweetpotato lines. Lower-case letters indicated significantly different at P<0.01. The three lines (L3, L8 and L11) with the highest expression level of IbMPK6 were marked with red border"

Fig. 2

Low temperature-resistance detection of IbMPK6-overexpressing sweetpotato A: Phenotype analysis; B: Optimal/maximal quantum yield of PSⅡ; C: MDA content. * and ** indicated significantly different at P<0.05 and P<0.01, respectively"

Fig. 3

Oxidative stress-resistance analysis of IbMPK6-overexprssion sweetpotato under low temperature treatment A: DAB staining; B: NBT staining; C: H2O2 content"

Fig. 4

Expression pattern of low temperature related genes"

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