二斑叶螨抗性木薯木质素合成途径基因表达特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0500

生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 161-171.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0500

二斑叶螨抗性木薯木质素合成途径基因表达特性研究

姚晓文¹^,²^,³(), 梁晓²^,³, 陈青²^,³(), 伍春玲²^,³, 刘迎²^,³, 刘小强²^,³, 税军¹^,²^,³, 乔阳¹^,²^,³, 毛奕茗¹, 陈银华¹, 张银东¹

1.海南大学热带作物学院，海口 571000
2.中国热带农业科学院环境与植物保护研究所农业农村部热带作物有害生物综合治理重点实验室海南省热带作物病虫害生物防治工程技术研究中心，海口 571101
3.中国热带农业科学院三亚研究院海南省南繁生物安全与分子育种重点实验室，三亚 572000

收稿日期:2022-04-22 出版日期:2023-02-26 发布日期:2023-03-07
作者简介:姚晓文，男，硕士，研究方向：抗虫种质资源挖掘与创新利用；E-mail: seanyao9712@163.com
基金资助:
国家重点研发计划项目(2020YFD1000603);国家木薯产业技术体系虫害防控岗位科学家专项(CARS-11-HNCQ);农业农村部农业资源调查与保护利用专项(NFZX-2021);海南省重大科技计划项目(ZDKJ202002)

Study on the Expression Pattern of Genes in Lignin Biosynthesis Pathway of Cassava Resisting to Tetranychus urticae

YAO Xiao-wen¹^,²^,³(), LIANG Xiao²^,³, CHEN Qing²^,³(), WU Chun-ling²^,³, LIU Ying²^,³, LIU Xiao-qiang²^,³, SHUI Jun¹^,²^,³, QIAO Yang¹^,²^,³, MAO Yi-ming¹, CHEN Yin-hua¹, ZHANG Yin-dong¹

1. College of Tropical Crops, Hainan University, Haikou 571000
2. Environment and Plant Protection Institute, China Academy of Tropical Agricultural Sciences, Key Laboratory of Integrated Pest Management on Tropical Crops, Ministry of Agriculture and Rural Affairs, Hainan Engineering Research Center for Biological Control of Tropical Crops Diseases and Insect Pests, Haikou 571101
3. Sanya Research Academy, Chinese Academy of Tropical Agriculture Science, Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Sanya 572000

Received:2022-04-22 Published:2023-02-26 Online:2023-03-07

摘要/Abstract

摘要：

木质素是作物防御有害生物的重要理化屏障，但其在木薯抗虫性中的作用机制尚不明确。为了探讨木质素合成途径基因在木薯抗二斑叶螨中的作用，以抗螨木薯品种C1115、缅甸（Myanmar）、SC9和感螨木薯品种SC205、面包（Bread）、BRA900为材料，采用实时荧光定量PCR分析二斑叶螨取食上述木薯品种不同时间后（0、1、4 d）木质素合成途径基因（PAL、4CL、C4H、HCT、CSE、COMT、CCoAOMT、F5H、CCR、CAD）在不同品种间的表达量差异。结果表明，与螨害前相比，抗、感螨木薯品种中C4H、HCT、CSE、F5H、CAD的表达量虽总体上有所提高，但并不是都能够达到显著差异水平，并且相同螨害时间内，这5个基因的表达量在抗、感木薯品种间整体上也未表现出显著差异。与之相反，PAL、4CL、CCoAOMT、CCR这4个基因的表达量在抗螨木薯品种中显著提高，在感螨木薯品种中显著降低或无显著差异，并且在相同的螨害时间内，抗螨木薯品种中这4个基因的表达量普遍高于感螨木薯品种，尤其以螨害4 d 后表达量表现出显著差异，进一步相关性分析发现，这4个基因表达量的提高与木薯抗螨性显著正相关。根据上述结果可以推测，螨害后木质素合成途径基因PAL、4CL、CCoAOMT、CCR表达量的高低可能与木薯品种的抗螨性水平有关。

关键词: 木薯, 二斑叶螨, 木质素合成途径, 基因表达, 抗、感螨木薯品种, 螨害时间, 抗性相关性

Abstract:

Lignin is an important physicochemical barrier for crops to defend against pests, however, its mechanism of cassava resistance to insect remains unclear. In order to explore the role of lignin biosynthesis pathway genes in cassava resisting to Tetranychus urticae（two-spotted spider mite, TSSM）, the mite-resistant cassava cultivar C1115, Myanmar, SC9 and mite-susceptible cassava cultivar SC205, Bread and BRA900 were applied during mite-cassava interaction. Furthermore, real-time quantitative PCR was used to analyze the expression pattern of lignin biosynthesis pathway genes（PAL, 4CL, C4H, HCT, CSE, COMT, CCoAOMT, F5H, CCR and CAD）among different cultivars infested by TSSM at different times of 0, 1, and 4 d. The results showed that the expressions of C4H, HCT, CSE, F5H and CAD in TSSM-infested mite-resistant and mite-susceptible cassava cultivars generally increased compared with those before infestation, but they did not always show significant differences, in addition, there was no significant difference in the expressions of these five genes among the mite-resistant and mite-susceptible cassava cultivars at the identical TSSM infested time. On the contrary, compared with those before infestation, the expressions of PAL, 4CL, CCoAOMT and CCR significantly increased in the TSSM infested mite-resistant cassava cultivars. However, the expressions of those four genes in mite-susceptible cultivars were either unchanged or significantly decreased, moreover, during the same mite-infested time, the expressions of these four genes in mite-resistant cassava cultivars were generally higher than those in mite-infested cassava cultivars, especially when the cassava plants were infested by TSSM at 4 d. Further correlation analysis found that the increase in the expressions of these four genes was significantly and positively correlated with the resistance of cassava to mite. To sum up, the above results speculated that the expressions of lignin biosynthesis pathway genes PAL, 4CL, CCoAOMT and CCR may be probably related to the resistance level of cassava cultivars.

Key words: cassava（Manihot esculenta Crantz）, Tetranychus urticae, lignin biosynthesis pathway, gene expression, mite-resistant and mite-susceptible cassava cultivars, mite infestation time, correlation of resistance

姚晓文, 梁晓, 陈青, 伍春玲, 刘迎, 刘小强, 税军, 乔阳, 毛奕茗, 陈银华, 张银东. 二斑叶螨抗性木薯木质素合成途径基因表达特性研究[J]. 生物技术通报, 2023, 39(2): 161-171.

YAO Xiao-wen, LIANG Xiao, CHEN Qing, WU Chun-ling, LIU Ying, LIU Xiao-qiang, SHUI Jun, QIAO Yang, MAO Yi-ming, CHEN Yin-hua, ZHANG Yin-dong. Study on the Expression Pattern of Genes in Lignin Biosynthesis Pathway of Cassava Resisting to Tetranychus urticae[J]. Biotechnology Bulletin, 2023, 39(2): 161-171.

图/表 11

表1 木薯木质素合成途径候选基因引物

Table 1 Candidate gene primers in the synthesis pathways of cassava（Manihot esculenta Crantz）lignin

Gene name	Gene ID	Primer sequence（5'-3'）	Tm/℃	Product size/bp
PAL	MANES_08G008400	F: AGGCATTTGGAGGAGAACTTGA R: TCGCAGAATCTTGATGGGTGA	61.2 59.8	113
C4H	MANES_18G126900	F: GCTCGCCAACAATCCTGCTC R: CTCCTCCTTCCAACTCCAAACG	58.7 59.3	158
4CL	MANES_17G006400	F: GGACTGTTGTCACTGGGCACTA R: TCTTCGTCAACGCTTGGAGTA	59.9 58.5	133
CCR	MANES_04G103200	F: GTCAATGCTGTTCAAGGCTATGTG R: AAATGTATCGCCCGGAGGC	61.6 62.2	94
CAD	MANES_13G117800	F: ACCAGAGGGAATGTCACCAGAA R: CCCTACTCCTCCAAGCCCTAAT	60.8 60.9	133
HCT	MANES_04G101700	F: GACCTTGTGGTGCCGAGATTC R: CGTCCAGCCATCGGATAGAAC	61.7 61.9	137
CSE	MANES_12G157800	F: TCCAATCGGAACCCAACACG R: AAGAGCAGCCCATACACGAAGAG	63.6 62.7	112
CCoAOMT	MANES_10G078800	F: GCCCACATCAATGGCTTCC R: GCTCCTTCATAGGTTCAGGCTCT	60.7 61.1	152
COMT	MANES_01G043700	F: GGCTGACCACTCAACCATTACC R: GACCGCTCCAGTTCCACCA	60.9 61.2	103
F5H	MANES_04G084300	F: GGTGGCATCGGCAATAGAGTG R: TGCAAGAGGAGTGGGATAGGTG	62.7 61.6	198

图1 螨害前后不同时间各品种PAL基因表达量变化 A：螨害前后各品种内基因表达量变化；B：螨害前后同一时间品种间基因表达量变化。不同小写字母表示螨害不同时间后表达量与螨害前差异显著（P<0.05）。下同

Fig. 1 Changes of PAL gene expressions in different varieties at different times before and after mite infestation A: Changes of gene expressions in various varieties before and after mite infestation. B: Changes of gene expressions between varieties at the same time before and after mite infestation. Different lowercase letters indicate that the expression amount after different time of mite infested is significantly different from that before mite infested（P<0.05）. The same below

图2 螨害前后不同时间各品种4CL基因表达量变化

Fig. 2 Changes of 4 CL gene expressions in different varieties at different times before and after mite infestation

图3 螨害前后不同时间各品种C4H基因表达量变化

Fig. 3 Changes of C4H gene expressions in different varieties at different times before and after mite infestation

图4 螨害前后不同时间各品种HCT基因表达量变化

Fig. 4 Changes of HCT gene expressions in different varieties at different times before and after mite infestation

图5 螨害前后不同时间各品种CSE基因表达量变化

Fig. 5 Changes of CSE gene expressions in different varieties at different times before and after mite infestation

图6 螨害前后不同时间各品种COMT基因表达量变化

Fig. 6 Changes of COMT gene expressions in different varieties at different times before and after mite infestation

图7 螨害前后不同时间各品种CCoAOMT基因表达量变化

Fig. 7 Changes of CCoAOMT gene expressions in different varieties at different times before and after mite inf-estation

图8 螨害前后不同时间各品种F5H基因表达量变化

Fig. 8 Changes of F5H gene expressions in different varieties at different times before and after mite infestation

图9 螨害前后不同时间各品种CCR基因表达量变化

Fig. 9 Changes of CCR gene expressions in different varieties at different times before and after mite infestation

图10 螨害前后不同时间各品种CAD基因表达量变化

Fig. 10 Changes of CAD gene expressions in different varieties at different times before and after mite infestation

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二斑叶螨抗性木薯木质素合成途径基因表达特性研究

Study on the Expression Pattern of Genes in Lignin Biosynthesis Pathway of Cassava Resisting to Tetranychus urticae

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