水稻优良三系不育系福农A多抗性基因分析及表达特性研究

连玲; 涂诗航; 张居念; 董萌; 何炜; 郑燕梅; 解振兴; 施龙清; 姜照伟; 吴春珠

水稻优良三系不育系福农A多抗性基因分析及表达特性研究

连玲^{1, 2,},
涂诗航¹,
张居念¹,
董萌¹,
何炜^{1, 2},
郑燕梅^{1, 2},
解振兴¹,
施龙清¹,
姜照伟¹,
吴春珠^1, ,

1.
福建省农业科学院水稻研究所，福建　福州　350019
2.
农业农村部华南杂交水稻种质创新与分子育种重点实验室/福州（国家）水稻改良分中心/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室/福建省作物分子育种省部共建国家重点实验室培育基地/水稻国家工程研究中心/杂交水稻国家重点实验室华南研究基地，福建　福州　350003

基金项目: 福建省科技计划公益类专项（2022R10230012）；福建省自然科学基金（2022J01449）；福建省农业科学院农业科技专项（GJYS202405）；福建省农业科学院科技创新团队建设项目（CXTD2021005-3）

详细信息

作者简介:
连玲（1983 —），女，硕士，助理研究员，主要从事水稻分子生物学与分子育种研究，E-mail：lianling51@163.com

通讯作者:
吴春珠（1971 —），女，副研究员，主要从事水稻遗传育种研究，E-mail: wuchunzhu@faas.cn

中图分类号: S511
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出版历程
- 收稿日期: 2024-06-05
- 修回日期: 2024-07-16
- 网络出版日期: 2024-10-31

Analysis of disease and insect resistance genes in excellent sterile line FunongA

LIAN Ling^{1, 2
,},
TU Shihang¹,
ZHANG Junian¹,
DONG Meng¹,
HE Wei^{1, 2},
ZHENG Yanmei^{1, 2},
XIE Zhenxing¹,
SHI Longqing¹,
JIANG Zhaowei¹,
WU Chunzhu^{1
, ,}

1.
Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350019, China
2.
Key Laboratory of Germplasm Innovation and Molecular Breeding of Hybrid Rice for South China, Ministry of Agriculture, China/Fuzhou Branch, National Rice Improvement Center of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding/Incubator of National Key Laboratory of Fujian Germplasm Innovation and Molecular Breeding between Fujian and Ministry of Sciences & Technology/National Engineering Laboratory of Rice/South Base of National Key Laboratory of Hybrid Rice of China, Fuzhou, Fujian　350003, China

摘要

摘要: 目的分析福农A中的抗病虫基因，为该不育系更好地应用于育种生产提供理论依据。方法以福农A及亲本福稻B、华航丝苗为材料，利用抗稻瘟病基因分子标记检测各材料中抗稻瘟病基因情况，并应用高密度芯片检测抗白叶枯病、抗病毒及抗褐飞虱基因情况。PCR扩增获得福农A及亲本福稻B、华航丝苗中的抗稻瘟病基因，进行序列比对分析。植株培养至三叶一心期，喷雾接稻瘟病菌并取样，采用 SYBR Green I 荧光定量 PCR（qRT-PCR）分析抗稻瘟病基因的表达模式。结果福农A及亲本福稻B、华航丝苗中均含有稻瘟病抗性基因Pib、Pia、Pid3和Pid2，但均不含Pi9、Pi54、Pigm、Pit、Pi2；另外，福农A和华航丝苗中含有Pi5，而福稻B中含有Pita和Pi37。福农A及亲本福稻B和华航丝苗中均含有抗白叶枯病基因Xa21和抗黄色斑驳病毒病基因Rymv1；福农A及华航丝苗中含持久性抗水稻条叶枯病毒基因STV11；但它们中均不含抗褐飞虱基因Bph14、Bph15、Bph18、 Bph26、 Bph6、 Bph9。扩增获得的 Pi5、Pia、Pib、Pid3、Pid2基因序列长分别为5 672、2 597、5 532、2 865、3847 bp；福农A与亲本华航丝苗中的Pi5基因序列完全一致；福农A和亲本华航丝苗、福稻B中的Pia、Pib基因序列均完全一致；福农A和华航丝苗中的Pid3和Pid2基因序列完全一致，而它们与福稻B的序列分别有2个和5个碱基差异。在福农A中，抗稻瘟病基因Pi5和Pib的表达明显受稻瘟病菌的诱导，接菌72 h时表达量最高；Pia和Pid3的表达随接菌时间的延长而逐渐升高，接菌96 h时表达量最高；Pid2的表达先升高后降低，且在接菌24 h时表达量最高。结论水稻三系不育系福农A中含有稻瘟病抗性基因Pi5、Pib、Pia、Pid3和Pid2，抗白叶枯病基因Xa21、抗黄色斑驳病毒病基因Rymv1及持久性抗水稻条叶枯病毒基因STV11。因此，该不育系有望应用于多抗基因的聚合育种研究。
- 福农A /
- 稻瘟病抗性基因 /
- 序列分析 /
- 基因表达分析
Abstract: Objective Analysis of disease and insect resistance genes in FunongA, a three-line indica cytoplasmic male sterile line, will provide an important theoretical basis for the application of the sterile line in breeding production. Method Genomic DNA of FunongA, FudaoB and Huahangsimiao was extracted by CTAB method, then blast resistance genes were detected with molecular markers. Resistance genes related to bacterial blight, viral diseases and brown planthopper were analyzed by high density chip detection. Blast resistance genes of FunongA, Huahangsimiao and FudaoB were obtained by PCR, then sequencing and comparative analysis were performed. The samples were collected at 0h, 24h, 48h, 72h and 96h after inoculation with blast fungus. Total RNA was extracted using the Trizol method, and the expressions of blast resistance genes analyzed by SYBR Green I fluorescent quantitative PCR (qRT-PCR). Result FunongA, FudaoB and Huahangsimiao contain blast resistance genes Pib, Pia, Pid3, Pid2, bacterial blight resistance gene Xa21 and yellow mottle virus resistance gene Rymv1. Furthermore, FunongA and Huahangsimiao contain blast resistance gene Pi5 and stripe virus resistance gene STV11, and FudaoB contain blast resistance genes Pita and Pi37. In addition, they do not contain brown planthopper resistance genes. The fragments of Pi5, Pia, Pib, Pid3, Pid2 were 5672 bp, 2597 bp, 5532 bp, 2865 bp, 3847 bp, respectively. The sequences of Pi5, Pid3 and Pid2 in FunongA were consistent with that in Huahangsimiao, while there were 2 and 5 bases different from Pid3 and Pid2 of Fudao B, respectively. In Funong A, the expression of rice blast resistance genes Pi5 and Pib was significantly induced by blast fungus, and the highest at 72 h after inoculation. The expression of Pia and Pid3 increased gradually with the prolongation of inoculation time, and the highest at 96 h. The expression of Pid2 increased firstly and then decreased, and the highest at 24 h. Conclusion Three-line indica cytoplasmic male sterile line FunongA contains blast resistance genes Pi5, Pib, Pia, Pid3 and Pid2, which significantly induced by blast fungus. Funong A also contains bacterial blight resistance gene Xa21, yellow mottle virus resistance gene Rymv1 and stripe virus resistance gene STV11.
- FunongA /
- blast resistance gene /
- sequential analysis /
- gene expression

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图 1 福农A及亲本的植株表型

Figure 1. Plant phenotype of FunongA and its parents

下载: 全尺寸图片幻灯片

图 2 稻瘟病抗性基因分析

M为Mark 1000 bp/2000 bp，CK1为阳性对照，CK2为阴性对照，HH为华航丝苗，FD为福稻B，FN为福农A。

Figure 2. Analysis of rice blast resistance genes

M: Mark 1000 bp/2000 bp, CK1: Positive control, CK2: Negative control, HH: Huahangsimiao, FD: FudaoB, FN: FunongA.

下载: 全尺寸图片幻灯片

图 3 稻瘟病抗性基因的PCR扩增

M为Mark 10000 bp，FN为福农A， HH为华航丝苗，FD为福稻B。

Figure 3. Amplification of rice blast resistance genes by PCR

M: Mark 10000 bp, FN: FunongA , HH: Huahangsimiao, FD: FudaoB.

下载: 全尺寸图片幻灯片

图 4 稻瘟病抗性基因Pid3和Pid2的序列比对

方框内为碱基差异位置。

Figure 4. Sequence alignment analysis of blast resistance genes Pid3 and Pid2

Position of base difference is showed in the box.

下载: 全尺寸图片幻灯片

图 5 稻瘟病抗性基因的表达分析

*表示差异显著（P≤0.05）, **表示差异较显著（P≤0.01）, ***表示差异极显著（P≤0.001）。

Figure 5. Expression analysis of rice blast resistance genes

*indicated significant difference (P≤0.05), **indicated extremely significant difference (P≤0.01), *** indicated very extremely significant difference (P≤0.001).

下载: 全尺寸图片幻灯片

表 1 PCR及qRT-PCR引物

Table 1. Primers for PCR and qRT-PCR

引物 Primers	引物序列（5′-3′） Primer sequence（5′-3′）	片段大小（抗/感） Fragment size（Resistance/Susceptibility）/bp	参考文献 Reference
Pibdom	F：GAACAATGCCCAAACTTGAGA	365 (R) /无 (S)	[9]
Pibdom	R：GGGTCCACATGTCAGTGAGC	365 (R) /无 (S)	[9]
Pi9	F：GCTGTGCTCCAAATGAGGAT	291 (R) / 397 (S)	[10]
Pi9	R：GCGATCTCACATCCTTTGCT	291 (R) / 397 (S)	[10]
Pi5	F：ATAGATCATGCGCCCTCTTG	206 (R)/307 (S)	[10]
Pi5	R：TCATACCCCATTCGGTCATT	206 (R)/307 (S)	[10]
Pi54	F：CAATCTCCAAAGTTTTCAGG	216 (R)/359 (S)	[11]
Pi54	R：GCTTCAATCACTGCTAGACC	216 (R)/359 (S)	[11]
Pita	F：CTCCCTTGTTCGGTCAAAAA	467 (R)/850 (S)	[10]
	YL155：AGCAGGTTATAAGCTAGGCC
	R：CCACATTGATGAAGCCCATA
Pia	F：GCGACTGACACTTTCAATAGC	175 (R)/206 (S)	[12]
Pia	R：CGGTAGAGCAATTTAGAAGCAG	175 (R)/206 (S)	[12]
Pigm	F：CAGAGCAGTAACAAACCCTA	750 (R)/1800 (S)	[13]
Pigm	R：TCCGCAAGATCAACATTC	750 (R)/1800 (S)	[13]
Pit	F：ATGATAACCTCATCCTCAATAAGT	733(R)/无 (S)	[14]
Pit	R：GTTGGAGCTACGGTTGTTCAG	733(R)/无 (S)	[14]
Pid3	F：TACTACTCATGGAAGCTAGTTCTC	BamHI, 148 (R)/ 178 (S)	[15]
Pid3	R：AGCACTTCTTGACTACTGTCTGCCT	BamHI, 148 (R)/ 178 (S)	[15]
Pi2	F：CAGCGATGGTATGAGCACAA	450 (R)/ 282 (S)	[10]
Pi2	R：CGTTCCTATACTGCCACATCG	450 (R)/ 282 (S)	[10]
Pi37	F：TCTTGAGGGTCCCAGTGTAC	1149(R)/无(S)	[16]
Pi37	R：CGAACAGTGGCTGGTATCTC	1149(R)/无(S)	[16]
Pid2	F：GCGTCGAAGATGTCCTGAAGCTCA	629(R)/无 (S)	[17]
Pid2	R：GGCAGTCGTATTGCTGTGAA	629(R)/无 (S)	[17]
Pi5-DNA	F: CCTTCTCTCTTCTCGTTCTCCTATTTCACA	5672
Pi5-DNA	R: CAGTTGGTTTCTTGTAGGATTACAGGTCAGT	5672
Pia-DNA	F：ACATTTTAGTCAACTCGCCTGTTCTTTT	2597
Pia-DNA	R：CAATCAGAAAGCAACAGGTCCAAATAAC	2597
Pib-DNA	F：ACAAAATCCATTCAAAAATAGAACAGAGCA	5532
Pib-DNA	R：CACAGCCCCTCGGGTCCACAT	5532
Pid3-DNA	F：AAGCGAGAAGGAAGTAACACCCAAGG	2865
Pid3-DNA	R：AAGAATGAATGTCCTGACTGAAACCAACT	2865
Pid2-DNA	F：CTGGGTTTGAAGAAAAGAATAAAGGGAGTG	3847
Pid2-DNA	R：TGAAAATGATACTGAAAGGTCTGATGAAAA	3847
Pi5-qPCR	F：TTCTGCTCGCTATCCAATCCAATG	173
Pi5-qPCR	R：TTTCTGTATGATGTTGTTTGCTTCTCCTC	173
Pia-qPCR	F：CCAGCCACTTGCTCTTGTTACCATAG	134
Pia-qPCR	R：GCCGCATCCTTTCAAGTGTTTTATCA	134
Pib-qPCR	F：GGACAAGTGTTGGTGCTTCGGAG	176
Pib-qPCR	R：CCTTGGGCTTTGATAAACACCGCT	176
Pid3-qPCR	F：AAGCAGTGGAGGTCGCCAAGTC	138
Pid3-qPCR	R：CGTCCTTGAACCGCTCGGCT	138
Pid2-qPCR	F：ACTAACTTCTTCCCTCCTGCGGCT	175
Pid2-qPCR	R：AAGATGCGAAACGAGTTGTATTCCC	175
Actin150	F：AGTGTCTGGATTGGAGGAT	147
Actin150	R：TCTTGGCTTAGCATTCTTG	147

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表 2 抗病虫基因分析结果

Table 2. Analysis of disease and insect resistance genes

基因名称 Genes	染色体 Chromosome	表型 Phenotype	探针类型 Probe type	福农A FunongA	福稻B FudaoB	华航丝苗 Huahangsimiao
xa13	8	白叶枯抗性	单倍型	0.08	0.08	0.22
Xa21	11	白叶枯抗性	单倍型	1.00	1.00	1.00
Xa23	11	白叶枯抗性	单倍型	0.07	0.10	0.07
xa5	5	白叶枯抗性	单倍型	0.35	0.31	0.35
Xa7	6	白叶枯抗性	单倍型	0.11	0.44	0.11
Rymv1	4	抗黄色斑驳病毒病	SNP	√	√	√
STV11	11	持久性抗水稻条叶枯病毒	SNP	√		√
Bph14	3	褐飞虱抗性	单倍型	0.48	0.55	0.34
Bph15	4	褐飞虱抗性	单倍型	0.16	0.16	0.16
Bph18	12	褐飞虱抗性	单倍型	0.69	0.59	0.69
Bph26	12	褐飞虱抗性	单倍型	0.52	0.43	0.52
Bph6	4	褐飞虱抗性	单倍型	0.62	0.62	0.62
Bph9	12	褐飞虱抗性	单倍型	0.40	0.28	0.40
表格中探针类型为“SNP”的基因，样品编号下方的“√”表示与代表品种基因型一致，空格表示未检测到相关基因；表格中探针类型为“单倍型”的基因，样品编号下方的数字表示与标准样对比的相似度，“1.00”表示与标准样相似度达100%，样品中存在该基因。 For genes with probe type "SNP" in the table, "√" indicated a consistent genotype with the reference variety and a blank indicated that the related gene was not detected. For genes with probe type "haplotype"，the number represented the similarity compared to reference sequence. "1.00" indicated a 100% similarity to the reference sequence, and the gene existed in the sample.

下载: 导出CSV

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