Expression and Adversity Resistance Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox

doi:10.16420/j.issn.0513-353x.2021-0987

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (2): 359-370.doi: 10.16420/j.issn.0513-353x.2021-0987

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Expression and Adversity Resistance Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox

REN Fei, LU Miaomiao, LIU Jixiang, CHEN Xinli, LIU Daofeng, SUI Shunzhao, MA Jing^*()

Chongqing Engineering Research Center for Floriculture，Key Laboratory of Horticulture Science for Southern Mountainous Regions，Ministry of Education，College of Horticulture and Landscape，Southwest University，Chongqing 400715，China

Received:2022-09-03 Revised:2022-10-27 Online:2023-02-25 Published:2023-03-06
Contact: *（E-mail：majing427@swu.edu.cn）

Abstract

Abstract:

To characterize the function of CpLEA，a group three LEA protein gene of Chimonanthus praecox（wintersweet），CpLEA is transformed into Escherichia coli and Arabidopsis thaliana. Compared with the control strain，transgenic E. coli showed higher tolerance to low-temperature. In vitro，purified CpLEA protein could protect lactate dehydrogenase（LDH）from inactivation. Overexpression of the CpLEA gene in Arabidopsis also improved the resistance of transgenic Arabidopsis to cold and drought stresses and the degree of improved resistance was positively correlated with the accumulation of gene overexpression. The expression characteristics of CpLEA gene were further analyzed by qRT-PCR in wintersweet cutflower branch during flower-bud and display-petal stage respectively under 4 ℃ low-temperature treatment. CpLEA gene expression was found to gradually increase after low-temperature treatment in cutflower branch at flower-bud stage，reaching a peak at 48 h of treatment，which was about 28 times higher than expression of the untreated group，and then the expression decreased，but was still higher than the untreated group. The expression of CpLEA gradually increased with the extension of low-temperature treatment time in cutflower branch at display-petal stage，then reached the highest level at 72 h，which was nine times as much as the untreated group. The expression of CpLEA in cutflowers remained basically constant at room temperature，while the low-temperature treatment induced a different degree of expression increase of CpLEA in the early stage of flower development. These results indicated that the CpLEA would play a protective role in plant cells under low-temperature stress，especially in early stage of flower development.

Key words: Chimonanthus praecox, LEA, prokaryotic expression, transgenic, low-temperature stress

CLC Number:

S685.99

REN Fei, LU Miaomiao, LIU Jixiang, CHEN Xinli, LIU Daofeng, SUI Shunzhao, MA Jing. Expression and Adversity Resistance Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox[J]. Acta Horticulturae Sinica, 2023, 50(2): 359-370.

Figures/Tables 10

Table 1 Primer sequences used in real-time quantitative PCR

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
CpLEA	CAGGAGAAGACGAGTCAGATGGT	CCGAGTCCCTTGTAGATTGG
Atactin	CTTCGTCTTCCACTTCAG	ATCATACCAGTCTCAACAC
Actin	GTTATGGTTGGGATGGGACAGAAAG	GGGCTTCAGTAAGGAAACAGGA
Tublin	TAGTGACAAGACAGTAGGTGGAGGT	GTAGGTTCCAGTCCTCACTTCATC

Fig. 1 SDS-PAGE electrophoresis of CpLEA protein obtained by induction of Escherichia coli BL21/pET32a（+）-CpLEA M：Protein marker；1：Total protein without induction；2：Total protein after induction；3：Supernatant protein after induction；4：Precipitation protein after induction；5：Buffering solution；6，7，8：20，50，300 mmol · L-1 imidazole elution.

Fig. 2 Colony density（A）and bacterial growth curve（B）of Escherichia coli to CpLEA strain BL21/pET32a（+）-CpLEA and to empty vector strain BL21/pET32a（+）

Fig. 3 Lactate dehydrogenase（LDH）activity by CpLEA protein under different stress conditions Different lowercase letters indicate significant differences among different reaction systems at 0.05 level.

Fig. 4 PCR（A1）and enzyme cutting verification（B1）electrophoresis of plant expression vector M：DL2000 marker；+：Positive controlar；-：Negative control，ddH2O.

Fig. 5 PCR（A）and RT-PCR（B）of Arabidopsis thaliana overexpressing CpLEA lines and expression analysis of CpLEA（C） M：DL2000 marker；1-11，L-1-L-9：Transgenic lines；+：Positive controlar；-：Negative control，ddH2O；WT：Wild type.

Fig. 6 Drought resistance in Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT） A：Seed germination under 10% PEG simulated drought；B：Seedling phenotype after drought stress；C：Survival rate after rewatering；D：CMS after drought stress. Different lowercase letters indicate significant differences among materials at 0.05 level.

Fig. 7 Phenotype of Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT）after cold treatment

Fig. 8 Cold resistance in Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT） Different lowercase letters indicate significant differences among different materials with the same treatment at 0.05 level.

Fig. 9 Expression analysis of CpLEA during flower stage Different lowercase letters indicate significant differences in the same treatment and different treatment times at 0.05 level.

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Expression and Adversity Resistance Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox

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