HIGS-SsCCS转基因拟南芥的菌核病抗性鉴定

doi:10.3864/j.issn.0578-1752.2020.04.008

摘要/Abstract

摘要：

【目的】菌核病是由核盘菌（Sclerotinia sclerotiorum）引起的一类真菌病害,核盘菌寄主范围广泛,严重危害多种作物的品质。本研究利用寄主诱导基因沉默（HIGS）的方法在寄主中诱导核盘菌致病相关基因的沉默从而增强寄主的菌核病抗性,为菌核病抗病育种提供新的思路。【方法】铜锌超氧化物歧化酶是一种重要的抗氧化剂,以核盘菌铜锌超氧化物歧化酶铜伴侣基因（copper chaperone for copper/zinc superoxide dismutase,SsCCS）为靶基因,通过生物信息学分析该基因的结构特点,并利用MEGA6.0软件构建系统发育树;通过分别比对拟南芥及核盘菌基因组,选择特异的干扰片段进行扩增;采用农杆菌介导的浸花序法,将HIGS载体转入拟南芥Col-0,通过DNA鉴定以及标记筛选出稳定的HIGS-CCS转基因拟南芥;选取4—5周龄的HIGS-CCS转基因拟南芥植株叶片接种核盘菌野生菌株1980,于接种24 h后统计病斑面积,分析转基因株系的菌核病抗性;通过qRT-PCR分析核盘菌侵染转基因植株过程中SsCCS的表达情况;同时在接种6、12、24 h后利用DAB染色的方法检测转基因植株与核盘菌互作过程中H₂O₂的积累。【结果】生物信息学分析结果表明,SsCCS（SS1G_00102）全长为1 010 bp,编码序列长759 bp,共编码253个氨基酸,该蛋白分子质量为27 176.96 Da,等电点（PI）为5.04,与灰霉病菌BcCCS（EDN25358）亲缘关系最近,氨基酸同源性达到87%,与拟南芥AtCCS（AT1G12520.1）亲缘关系较远;通过与核盘菌以及拟南芥基因组比对,选择314 bp特异干扰片段,成功构建SsCCS的HIGS表达载体,转化拟南芥。T₁及T₂代转基因拟南芥接种核盘菌24 h后的病斑面积均小于野生型拟南芥。从T₂代中获得3个稳定表达的T₃代HIGS-CCS转基因拟南芥株系：HIGS-CCS-5、HIGS-CCS-8、HIGS-CCS-13;与野生型拟南芥相比,转基因拟南芥在接种核盘菌24 h后,病斑面积显著减小46%—61%;qRT-PCR结果显示核盘菌在侵染转基因植株过程中,SsCCS的表达量显著低于侵染野生型拟南芥。DAB染色结果表明,在侵染过程中,转基因拟南芥植株中H₂O₂的积累明显少于野生型拟南芥,ROS水平降低。【结论】利用HIGS方法在拟南芥中干扰核盘菌SsCCS的表达能够显著提高拟南芥的抗病性,研究结果为油菜等农作物菌核病抗病改良提供了参考。

关键词: 拟南芥, 寄主诱导的基因沉默, 核盘菌, SsCCS, 抗病性, 菌核病

Abstract:

【Objective】Sclerotinia stem rot is a kind of fungal disease caused by Sclerotinia sclerotiorum. The host range of S. sclerotiorum is wide, which seriously endangers the quality of many crops. The objective of this study is to enhance the resistance to stem rot by silencing the virulence genes of S. sclerotiorum in host via the host-induced gene silencing (HIGS) technology, and to provide new ideas for breeding of sclerotinia stem rot resistance.【Method】The gene encoding a copper chaperone for copper/zinc superoxide dismutase of S. sclerotiorum (SsCCS) was selected as the target gene, the sequences were analyzed by bioinformatics tools, and the phylogenetic tree was constructed using MEGA6.0 software. The specific interference fragment was selected for amplification after comparing the genome of Arabidopsis thaliana and S. sclerotiorum, respectively. HIGS vector containing the RNAi structure of SsCCS was transferred into wild type A. thaliana Col-0 mediated by Agrobacterium, and the stable HIGS-CCS transgenic A. thaliana lines were screened by DNA identification and labeling. The leaves of HIGS-CCS transgenic plants grown for 4-5 weeks were selected to analyze the resistance to sclerotinia stem rot according to the lesion area at 24 h after inoculated with S. sclerotiorum. The relative expression level of SsCCS during infecting was analyzed by qRT-PCR. The accumulation of H₂O₂ during the period of interaction between transgenic plants and S. sclerotiorum was detected by DAB staining at 6, 12 and 24 hpi.【Result】Bioinformatics analysis showed that the length of genome sequence of SsCCS (SS1G_00102) is 1 010 bp, while the length of its coding sequence (CDS) is 759 bp, encoding a protein with 253 amino acids, the molecular weight is 2 7176.96 Da, the isoelectric point (PI) is 5.04. SsCCS has 87% amino acid homology to BcCCS (EDN25358) while far to AtCCS (AT1G12520.1). By aligning with the genome of S. sclerotiorum and A. thaliana, a 314 bp specific interference fragment was selected and constructed the HIGS vector successfully and transformed into A. thaliana. The lesion of T₁ and T₂ generation transgenic lines was smaller than that of wild type A. thaliana 24 h after inoculated with strain 1980. From the T₂ generation, three stably expressed T₃ generation HIGS-CCS transgenic A. thaliana lines (HIGS-CCS-5, HIGS-CCS-8, HIGS-CCS-13) were obtained. Compared with wild type A. thaliana, the lesion area on the HIGS-CCS transgenic plants was reduced by 46% to 61% 24 h after inoculated with strain 1980. The expression of SsCCS was significantly reduced by 98% in the HIGS-CCS transgenic plants compared to that in wild type A. thaliana at 6 h after inoculated with S. sclerotiorum strain 1980. Furthermore, the accumulation of H₂O₂was decreased in transgenic plants as revealed by DAB staining, indicating the ROS production was reduced in transgenic plants.【Conclusion】The resistance to stem rot can be significantly enhanced by silencing the SsCCS of S. sclerotiorum in A. thaliana via the HIGS. This study provides a reference for the resistance improvement to sclerotinia stem rot of host crops, such as rapeseed.

Key words: Arabidopsis thaliana, host-induced gene silencing (HIGS), Sclerotinia sclerotiorum, SsCCS, disease resistance, sclerotinia stem rot

柴亚茹,丁一娟,周思钰,杨文静,闫宝琴,远俊虎,钱伟. HIGS-SsCCS转基因拟南芥的菌核病抗性鉴定[J]. 中国农业科学, 2020, 53(4): 761-770.

YaRu CHAI,YiJuan DING,SiYu ZHOU,WenJing YANG,BaoQin YAN,JunHu YUAN,Wei QIAN. Identification of the Resistance to Sclerotinia Stem Rot in HIGS-SsCCS Transgenic Arabidopsis thaliana[J]. Scientia Agricultura Sinica, 2020, 53(4): 761-770.

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引物名称 Primer name	引物序列 Primer sequence
SsCCS-SF	CCC AAGCTT GAATTC TATCTACGGCTGGTTTATGG
SsCCS-SR	TAA GATATC GCATACCTTTGCTAATCTCATCC
SsCCS-AF	CGC GGATCC CTCGAG TATCTACGGCTGGTTTATGG
SsCCS-AR	TAA CTGCAG GCATACCTTTGCTAATCTCATCC
SsTubF	GTGAGGCTGAGGGCTGTGA
SsTubR	CCTTTGGCGATGGGACG
RT-SsCCSF	TCCGTGTTCCCCTTTATCCT
RT-SsCCSR	CCTCAATCGCCCTTACAATCT
PIF	GCCCTTCCTCCCTTTATTTC
PIR	GAGCTGACATCGACACCAAC
M13F	TTGTAAAACGACGGCCAG
M13R	ACACAGGAAACAGCTATGAC
RVF	CGCACAATCCCACTATCCTT
RVR	AAAAGACAAAAGTGGGGTAG

编号 Code	T₁代 T₁generation				T₂代病斑面积 T₂ generation lesion area (cm²)
编号 Code	阳性种子数 Positive	非阳性种子数 Negative	分离比 Segregation ratio	病斑面积 Lesion area (cm²)	T₂代病斑面积 T₂ generation lesion area (cm²)
WT	-	-	-	1.46±0.26a	1.02±0.36a
HIGS-CCS-13	73	27	3﹕1	0.69±0.2b	0.79±0.27b
HIGS-CCS-8	69	31	3﹕1	0.71±0.17b	0.48±0.21b
HIGS-CCS-5	78	22	3﹕1	0.89±0.13b	0.55±0.23b
HIGS-CCS-14	79	21	3﹕1	0.82±0.27b	0.93±0.27a
HIGS-CCS-7	92	8	15﹕1	0.77±0.18b
HIGS-CCS-18	69	31	3﹕1	0.91±0.53a
HIGS-CCS-22	85	25	3﹕1	1.11±0.08a
HIGS-CCS-3	71	29	3﹕1	1.11±0.25a
HIGS-CCS-19	44	56	1﹕1	1.12±0.11a
HIGS-CCS-4	56	44	1﹕1	1.15±0.13a
HIGS-CCS-17	100	0	-	1.27±0.28a
HIGS-CCS-9	78	22	3﹕1	1.37±0.18a
HIGS-CCS-11	95	5	15﹕1	1.21±0.24a
HIGS-CCS-2	70	30	3﹕1	0.91
HIGS-CCS-20	76	34	3﹕1	1.02
HIGS-CCS-1	89	11	15﹕1	1.13
HIGS-CCS-12	74	26	3﹕1	1.26