利用TRV-HIGS技术鉴定核盘菌致病相关的分泌蛋白基因

doi:10.3864/j.issn.0578-1752.2019.23.008

Abstract

Abstract:

【Objective】 Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is the main problem in rapeseed planting in China, which causes serious yield and quality loss. Secretory proteins play an important role in the pathogenesis of pathogens. The genome of S. sclerotiorum contains a large number of genes encoding secretory proteins. The objective of this study is to identify and screen the secretory protein genes related to pathogenicity, reveal the pathogenic mechanism of S. sclerotiorum, and to provide an important target for the prevention and control of SSR. 【Method】 SMART software was used to analyze the protein domains of 8 candidate genes with signal peptides that were differentially expressed in the process of S. sclerotiorum infecting the susceptible and resistant Brassica oleracea lines, then the domains obtained by SMART analysis were annotated in SCOP, Pfam and PDB databases. The fragment with the length of around 300 bp in the encoding region of these genes was cloned into pTRV2 vector together with the GFP fragment. The suspension of pTRV1 was mixed equally with pTRV2-Gene and pTRV2-GFP, respectively. After 3 hours at room temperature, pTRV2-Gene vector and control (pTRV2-GFP) were transformed into 5-6 week-old leaves of Nicotiana benthamiana using syringe infiltration method. Subsequently, the infiltrated plants were cultured in dark for 48 hours and then grown in the normal light for 7 days. PDA mycelium blocks of S. sclerotiorum with a diameter of 6 mm were used to inoculate the infiltrated leaves of tobacco at the 9th day after transformation in vivo, in which the carrying surface was close to the leaves. After 48 hours of inoculation, the lesion size was measured and RNA from necrotic and infected tissues (around 1 cm from the edge of necrotic tissue) was extracted. qRT-PCR analysis was carried out to estimate the relative expression of target gene in N. benthamiana lines carrying TRV-HIGS vector. 【Result】 The putative functions of these 8 genes predicated with SMART and domain annotation were involved in the hydrolysis of proteins, nucleic acids or polysaccharides, the immunity response of host plants, and the tolerance to drugs and biotin synthesis of S. sclerotiorum. The average lesion area of control carrying TRV-GFP was 3.44 cm ² at 48 hours post inoculation of S. sclerotiorum. Except for one line (SS1G_07655), the lesion area of other 7 lines carrying TRV-HIGS vector was significantly lower than that of the control plants (P≤0.05), ranging from 1.63 to 2.61 cm ². qRT-PCR analysis showed that the gene expression level of these 7 genes in the TRV-HIGS lines was significantly lower than that of the control (P≤0.05). 【Conclusion】 Eight secretory protein genes with unknown function in S. sclerotiorum were successfully identified by TRV-HIGS technique. Seven genes related to the pathogenicity of S. sclerotiorum were screened out. Among them, SS1G_03146 with the strongest effect on the pathogenicity of S. sclerotiorum may be involved in the synthesis of biotin, and SS1G_04343 and SS1G_11912 may be involved in the immune response of host.

Key words: Sclerotinia sclerotiorum, TRV-HIGS, Nicotiana benthamiana, secretory protein, pathogenicity, qRT-PCR

YUAN JunHu,DING YiJuan,YANG WenJing,YAN BaoQin,CHAI YaRu,MEI JiaQin,QIAN Wei. Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS[J].Scientia Agricultura Sinica, 2019, 52(23): 4274-4284.

Figures/Tables 7

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基因 Gene	引物序列 Primer sequence (5′-3′)	产物大小 Product size (bp)	用途 Usage
SS1G_00263	F: CCGGAATTCCGGCAGCGCCTCAAGCTCGACTCAAATC R: CGAGCTCGCTGACGGTAGCGGAACCAACAACGG qF: TCTTTGAGGATGGAACTTGGAC qR: AGCCTGGCAAGCATAATCG	273	基因扩增Gene amplification qRT-PCR
SS1G_04945	F: CCGGAATTCCGGTCTCCTTCCTTCTCGGC R: CGAGCTCGCTCGTAATCGGCACCAT qF: TCTCAACGGTGCTCTTTACTTC qR: TTGCTATCAGGGACCCATCC	263	基因扩增Gene amplification qRT-PCR
SS1G_03181	F: CCGGAATTCCGGTGAGCGATGCTACCAACAGCGCCTAC R: CGAGCTCGCACCAGCACTTTGGAGAGCACCGTAA qF: TGCCGATTACGAGGGAACA qR: TGGAAACATCGACGGTGAAG	210	基因扩增Gene amplification qRT-PCR
SS1G_04343	F: CCGGAATTCCGGTGGTCTTTACGCTGGGTATTTC R: CGAGCTCGCACCGTTGGTTGTGTTTTCATT qF: TGGTCTTTACGCTGGGTATTTC qR: CAGACACTTGCGAATGGAGC	284	基因扩增Gene amplification qRT-PCR
SS1G_03146	F: CCGGAATTCCGGACATTTCCTCTTGAACCATCCCGTA R: CGAGCTCGTTTATGACACCCTTGTTTCCAGCGA qF: GCACATTTCCTCTTGAACCATC qR: GCAGTGTCACTTCCCACCATT	309	基因扩增Gene amplification qRT-PCR
SS1G_02250	F: CCGGAATTCCGGTCACACTTTTGGCATT R: CGAGCTCGATCAGCACGTTTTTCT qF: AAGCCAACACCAACCTCATC qR: CACTGGAGCGTAGTTCTCGTAG	272	基因扩增Gene amplification qRT-PCR
SS1G_11912	F: CCGGAATTCCGGTCAGCAGCTCCACCTCCACCACC R: CGAGCTCGATTCGCCTTTCCAAAATCCGTAT qF: TTCCCGAAACCGTTCCTAGT qR: TCACCATTGCTACTGCCACTT	257	基因扩增Gene amplification qRT-PCR
SS1G_07655 Sstub1 （内参基因Actin gene）	F: CCGGAATTCCGGGCTACTGTTCCTTTGGACTACGCT R: CGAGCTCGTTACTGAGGAGTGAGTCGTGTCGG qF: AATATGCCAGAGCCATCACA qR: CAGCGTAGTCCAAAGGAACAG qF: GTGAGGCTGAGGGCTGTGTGA qR: CCTTTGGCGATGGGACG	322	基因扩增Gene amplification qRT-PCR qRT-PCR

基因 Gene	结构域名称 Domain name	结构域位置 Location (aa)	E期望值 E-value	可能的功能或特性 Putative function or feature
SS1G_00263	SCOP:d1hw1a2	14-79	0.77	调节药物的耐受性 Regulation of drug tolerance
SS1G_04945	Pfam:Glyco_hydro_7	21-446	2.6e-200	O型糖苷水解酶 O-glycoside hydrolase
	PDB:3PL3\|A	19-446	0	纤维二糖水解酶 Cellobiohydrolase
	SCOP:d1gpia_	19-450	0	伴刀豆球蛋白A样凝集素/葡聚糖酶 Concanavalin A/Glucanase
SS1G_03181	Pfam:ASP	91-399	3.2e-69	天冬氨酰蛋白酶（酸性蛋白酶）Aspartyl proteases (acid proteases)
	SCOP:d1bxoa_	79-399	1e-81	酸性蛋白酶 Acid proteases
	PDB:3APP\|A	83-398	1e-90	酸性蛋白酶 Acid proteases
SS1G_04343	SCOP:d1gh8a_	339-377	0.39	翻译延长因子 Translation elongation factor
SS1G_04343	SCOP:d1fuia2	411-457	0.68	异构酶 Isomerase
SS1G_03146	SCOP:d1h4vb2	18-62	1.8	生物素合成酶 Biotin synthetase
SS1G_02250	SCOP:d1ed8a_	43-90	5.7	碱性磷酸酶 Alkaline phosphatase
SS1G_02250	SCOP:d1dofa_	215-241	0.86	L型天冬氨酸 L-aspartic acid
SS1G_11912	Pfam:NPP1	52-242	4.5e-61	坏死诱导蛋白 Necrosis inducing protein
	PDB:3GNZ\|P	32-242	4e-65	毒素 Toxin
	SCOP:d1fuia2	188-240	0.58	异构酶 Isomerase
SS1G_07655	Pro-kuma_activ	35-181	3.25e-19	肽酶 Peptidase
	PDB:3EDY\|A	35-581	2e-67	肽酶 Peptidase
	SCOP:d1gt91_	36-581	9e-19	枯草杆菌蛋白酶类 Subtilisins

Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS

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