CsEXPA8过表达对‘晚锦橙’生长及溃疡病抗性的影响

doi:10.16420/j.issn.0513-353x.2023-0145

摘要/Abstract

摘要：

为了明确扩展蛋白基因CsEXPA8的生物学功能，通过构建超量表达和干扰表达CsEXPA8的载体转化易感溃疡病的柑橘品种‘晚锦橙’，对获得的转基因植株进行表型分析和对溃疡病抗性评价。结果表明，CsEXPA8在‘晚锦橙’中具有组织表达特异性，并受溃疡病菌诱导表达；CsEXPA8的过表达促进了叶片的长度和宽度的增加以及春梢直径的增粗，提高了对溃疡病的敏感性；而干扰CsEXPA8的表达后，转基因植株的叶片长度、宽度和春梢直径变小，但对溃疡病的抗性增强。上述结果表明，CsEXPA8正向调节了‘晚锦橙’的生长，负向调控了‘晚锦橙’对溃疡病的抗性。

关键词: 柑橘, 扩展蛋白, CsEXPA8, 植物生长, 柑橘溃疡病, 抗性

Abstract:

To clarify the biological function of the expansion protein gene CsEXPA8，the vectors which excessed and inhibited the expression level of CsEXPA8 were constructed and then introduced in‘Wanjincheng’orange，respectively. The phenotype of transgenic plants was observed and their resistance to citrus canker was evaluated. The results showed that CsEXPA8 has tissue specific expression characteristics and its expression level in‘Wanjincheng’orange could be induced by Xanthomonas citri pv. citri，and that overexpression of CsEXPA8 could promote the growth of leaf length and width，and spring shoot，but also increase the sensitivity of transgenic plants to citrus canker，and that inhibiting the expression level of CsEXPA8 reduced the leaf length and width，and spring shoot，and enhanced the resistance of transgenic plants against citrus canker. These results indicated that CsEXPA8 positively and negatively regulates growth and resistance to citrus canker of‘Wanjincheng’orange，respectively.

Key words: citrus, expansins, CsEXPA8, plant growth, citrus canker, resistance

彭爱红, 张婧芸, 陈志毅, 苏娟, 何永睿, 姚利晓. CsEXPA8过表达对‘晚锦橙’生长及溃疡病抗性的影响[J]. 园艺学报, 2024, 51(5): 971-981.

PENG Aihong, ZHANG Jingyun, CHEN Zhiyi, SU Juan, HE Yongrui, YAO Lixiao. Effects of Overexpression of CsEXPA8 on Growth and Canker Disease Resistance in‘Wanjincheng’Orange[J]. Acta Horticulturae Sinica, 2024, 51(5): 971-981.

图/表 8

表1 本研究中所用引物序列

Table 1 The sequences of primer used in this study

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
CsEXPA8克隆	CsEXPA8-F	ATGGCCAATATTGCAGCATCATCTA
Clone of CsEXPA8	CsEXPA8-R	TAGATGATGCTGCAATATTGGCCAT
CsEXPA8 CDS克隆	pCsEXPA8-F	ggtaccATGGCCAATATTGCAGCATCAT
Clone of CDS of CsEXPA8	pCsEXPA8-R	gaattcCTAAGCTTTGAGCAATTCCTT
检测柑橘Actin的表达量	Actin-F	CATCCCTCAGCACCTTCC
Expression analysis of citrus Actin	Actin-R	CCAACCTTAGCACTTCTCC
检测柑橘CsEXPA8的表达量	qCsEXPA8-F	TGCTTTCGTGGCACATTTGG
Expression analysis of citrus CsEXPA8	qCsEXPA8-R	ACCCCCGCCATAGAAAGTTG
克隆CsEXPA8干扰片段	rCsEXPA8-F	ggatccatttaaatCCTTATTCCTCCTTTCTTCGC
Clone of RNAi fragment of CsEXPA8	rCsEXPA8-R	gtcgacggcgcgccGGCGGGGGTGATGCTTCCGG
检测GUS基因	GUS-F	CATGAAGATGCGGACTTGCG
Detection of GUS gene	GUS-R	GCTAACGTATCCACGCCGTA

图1 柑橘CsEXPA8表达的组织特异性（A）和受溃疡病菌的诱导表达（B）不同小写字母表示0.05水平差异显著。下同。

Fig. 1 Tissue specific（A）and induced by Xanthomonas citri pv. citri（B）expression analysis of CsEXPA8 Different lowercase letters indicate significant difference at 0.05 level. The same below.

图2 柑橘CsEXPA8与拟南芥（At）中扩展蛋白的进化树

Fig. 2 Evolutionary tree of CsEXPA8 and expansins in Arabidopsis thaliana（At）

图3 柑橘CsEXPA8基因的结构（A）、CDS序列（B）和编码蛋白的结构域（C）

Fig. 3 Structure（A）and CDS sequence（B）of CsEXPA8 gene and domain（C）of its encoded protein in citrus

图4 CsEXPA8过表达（A、C、E）和干扰表达（B、D、F）柑橘转基因植株的检测 M：Marker；WT：野生型；P：质体；p-：过表达植株；RNAi-：干扰表达植株。下同。

Fig. 4 Detection of overexpression（A，C，E）and interference expression（B，D，F）of CsEXPA8 in transgenic citrus plants M：Marker；WT：Wild-type；P：Plasmid；p-：Overexpressed plants；RNAi-：Interfering expressed plants. The same below.

图5 柑橘CsEXPA8超量表达（p-）和干扰表达（RNAi-）植株的表型分析

Fig. 5 Phenotypic analysis of CsEXPA8 overexpression（p-）and interference expression（RNAi-）plants in citrus

图6 转基因植株接种溃疡病菌后的发病情况

Fig. 6 Development of transgenic plants after inoculation with Xanthomonas citri pv. citri

图7 转基因植株对溃疡病的抗性评价

Fig. 7 Resistant evaluation of transgenic plants to citrus canker

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