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

Abstract

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

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

Identification of the Resistance to Sclerotinia Stem Rot in HIGS-SsCCS Transgenic Arabidopsis thaliana

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