利用BSA-Seq方法定位一个水稻早衰相关基因<i>OsBRCA</i>1

蒋家焕; 朱永生; 陈丽萍; 郑燕梅; 蔡秋华; 谢华安; 王爱荣; 张建福

doi:10.19303/j.issn.1008-0384.2022.002.001

利用BSA-Seq方法定位一个水稻早衰相关基因OsBRCA1

doi: 10.19303/j.issn.1008-0384.2022.002.001

1.
福建省农业科学院水稻研究所/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　福州　350003
2.
福建农林大学植物保护学院，福建　福州　350002

基金项目: 福建省自然科学基金项目（2018J01039、2020J011352）

详细信息

作者简介:
蒋家焕（1972−），男，副研究员，主要从事水稻遗传育种研究（E-mail：zysfaas@qq.com）

通讯作者:
张建福（1971−），男，研究员，博士生导师，主要从事水稻分子育种与分子生物学研究（E-mail：jianfzhang@163.com）

中图分类号: S 511
计量
- 文章访问数: 921
- HTML全文浏览量: 163
- PDF下载量: 61
- 被引次数: 0
出版历程
- 收稿日期: 2022-01-09
- 修回日期: 2022-02-10
- 网络出版日期: 2022-03-21
- 刊出日期: 2022-02-25

Mapping of Early Senescence-related OsBRCA1 in Rice by BSA-seq Technique

1.
Rice Research Institute, Fujian Academy of Agricultural Sciences/Key Laboratory of Hybrid Rice Germplasm Enhancement and Molecular Breeding in South China, Ministry of Agriculture, P.R. China/Fuzhou branch, National Rice Improvement Center of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding, Fuzhou, Fujian　350003, China
2.
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的水稻早衰突变体是研究水稻衰老机制的良好载体。定位和克隆水稻早衰相关基因，有助于理解水稻早衰的遗传规律和分子机制，为相关基因的作用机制研究奠定基础。方法用EMS化学诱变处理粳稻云引，获得一个稳定遗传的早衰突变体w14，与日本晴杂交构建F₂分离群体，并在群体中各选择100个隐性单株和显性单株的DNA等量混合，利用BSA-seq分析两个DNA池之间的差异位点，定位水稻早衰相关基因。结果突变体w14的早衰表型受单隐性基因控制，对两个DNA池测序结果进行单基因定位和突变基因的鉴定，发现单基因主峰位于Chr3，候选区间为 Chr3：27.5～29.5 Mb，进一步在目标区域内找到2个符合条件的候选因果变异。结论候选基因 LOC_Os03g49210突变位点是由野生型的C突变为T，且该突变位于候选基因的第2 外显子上，属于错义突变，造成了该基因编码的第20 个氨基酸由T（苏氨酸）变成I（异亮氨酸），从而可能导致了基因功能的改变，因此，将该基因定为本研究的候选基因，因与人类BRCA1同源，命名为 OsBRCA1。
- 水稻 /
- 突变体 /
- 早衰 /
- 基因克隆 /
- BSA-seq
Abstract: Objective Molecular mechanism of rice senescence was studied by locating and cloning the specific gene from a mutant artificially induced with early senescence. Methods Japonica rice Yunyin was treated with EMS to artificially induce a genetically stable early senescence w14. The mutant was crossed with Nipponbare to construct an isolated F₂ population. Subsequently, equal parts of the DNA of 100 recessive and dominant plants in the population were mixed. The differential loci between these two DNA pools were analyzed by BSA-seq to locate the target early senescence-related gene. Results The early senescence phenotype of w14 was found to be controlled by a single recessive gene. The mutagenic gene mapped and identified by the specific sequence showed the main peak located in Chr 3 and the candidate interval Chr 3: 27.5–29.5 Mb. In the region, two qualified candidate causal variants were found. Conclusion The mutation site of the candidate gene Loc_ Os03g49210 located in the 2^nd exon was determined to have changed in the wild-type from C to T. It was a non-synonymous mutation that resulted in the 20^th amino acid encoded by the gene changed from T (threonine) to I (isoleucine) and led to the functional alternation. Being homologous with human BRCA1, the target gene was named OsBRCA1.
- rice /
- mutant /
- early senescence /
- gene clone /
- BSA-seq

HTML全文

图 1 突变体 w14 测序数据的质量评估

Figure 1. Statistical analysis on sequencing data and quality on w14

下载: 全尺寸图片幻灯片

图 2 基因池间等位频率分析寻找目标基因的主峰位置

注：A：隐性纯合池；B：显性杂合池；C：两个混池之间的差异；D：目标基因的基因主峰。

Figure 2. Allele frequency between gene pools for locating main peak in target gene

Note: A: recessive homozygous pool; B: dominant heterozygous pool; C: difference between two mixed pools; D: main peak in target gene.

下载: 全尺寸图片幻灯片

图 3 测序结果中3号染色体的AF及AFD拟合曲线

Figure 3. AF and AFD fitting curves of Chr 3 in sequencing

下载: 全尺寸图片幻灯片

图 4 OsBRCA1在野生型和突变体w14的序列比对分析

注：A：基因组序列比对结果；B：氨基酸序列比对结果。

Figure 4. Sequence alignment between OsBRCA1 in wild-type and mutant w14

Note: A: genome sequence alignment; B: amino acid sequence alignment.

下载: 全尺寸图片幻灯片

表 1 用于 OsBRCA1基因组全长分段扩增的引物序列

Table 1. Primer sequences for genome full-length fragment amplification on OsBRCA1

序号 Number	标记 Marker	上游引物 Forward primer（5′-3′）	下游引物 Reverse primer（5′-3′）
1	49210-1	AATACCATATCGCCGTTTTCTT	AACTTTTCCATCACATCGTTCTAA
2	49210-2	TTGCCCGCATAATGTGACTG	AAGACTGGAGACTTGGGAGGTG
3	49210-3	TTTGCTATGCTGAACTCCCG	TCCCTTCCACCTTCTTCTTTG
4	49210-4	GATATGTTGGGTCATTTTGGAGC	TCAAGTTTGTAAGTTGGTGGTCG
5	49210-5	CAAGGTCCACAGGAAGAAGGT	AAGATTGTTGCACTCAAAGAAATG
6	49210-6	CTGGCTAACTCCACATGCTCTT	TTCCACTGGCCTACCTCACG
7	49210-7	CCAACTAATGAACCTGGAGCG	CAATATGTGCAACCAAAATGTGAA
8	49210-8	GGGCAGCCTTCACTAATGACA	TTACGCTGAAACAGGGAAATG
9	49210-9	GCTGAGGATGGAATACGAAGGA	GGTGGAGGGAAATCGAGGAG

下载: 导出CSV

表 2 测序数据和测序质量的统计分析

Table 2. Statistical analysis on sequencing data and quality

样本 Sample	原始数据 Raw base/bp	过滤数据 Clean base/bp	比例 Rate/%	Q20/%	Q30/%	GC/%
w14	7 057 752 750	6 731 469 250	95.38	96.10	91.15	44.27
隐性基因池 Recessive gene pool	12 841 555 750	12 418 982 750	96.71	96.00	91.04	44.43
显性基因池 Dominant gene pool	9 239 019 250	8 888 096 500	96.20	95.96	91.00	42.56
注： Q20及Q30：Phred数值大于20、30的碱基占总体碱基的百分比，其中，Phred=-10log₁₀(e)，e为错误率。 Note: Q20 and Q30: percentages of bases with Phred values greater than 20 and 30, respectively, in total number of bases; Phred=-10log₁₀(e); e: error rate.

下载: 导出CSV

表 3 目标染色体的因果变异鉴定结果

Table 3. Causal variant identification on target chromosome

染色体 Chromosome	物理位置 Physical location	野生型 Wild type	隐性池 Hidden pool	显性池 Dominant pool	野生型参考基因组 WT-IRGSP5
Chr3	27726166	1/1∶8∶1∶7	1/1∶41∶0∶41	0/1∶25∶19∶6	0/0∶1∶1∶0
Chr3	28129631	0/1∶11∶2∶9	0/1∶39∶2∶37	0/1∶31∶20∶11	0/0∶13∶13∶0
注：表中数据0/0表示参考基因组序列的纯合，1/1表示变异序列的纯合。 Note: * 0/0 and 1/1 represent homozygous of reference and variant genome sequence, respectively.

下载: 导出CSV

表 4 利用snpEff对候选因果变异进行注释的结果

Table 4. Annotated candidate causal variant by snpEff

差异位点　　　 Variable site	差异位点1　　　 Variable site 1	差异位点2　　　 Variable site 2
物理位置 Physical location	27726166	28129631
突变基因 Mutant gene	LOC_Os03g48626	LOC_Os03g49210
变异结果 Consequence	变异发生在内含子中	错义突变aCc(T) → aTc(I)由于C变异为T，导致第20个氨基酸T变为I
基因功能注释 Functional annotation	表达蛋白	含有BRCA1 C末端结构域的蛋白质

下载: 导出CSV

参考文献(19)

[1]	LIM P O, KIM H J, NAM H G. Leaf senescence [J]. Annual Review of Plant Biology, 2007, 58: 115−136. doi: 10.1146/annurev.arplant.57.032905.105316
[2]	BECK C I, ULRICH T H. Environmental release permits [J]. Bio/Technology, 1993, 11(12): 1524−1528.
[3]	SWAMINATHAN M S. Towards a hunger-free India [J]. The Indian Journal of Nutrition and Dietetics, 1999, 36(4): 108−117.
[4]	DAI L Y, LIU X L, XIAO Y H, et al. Recent advances in cloning and characterization of disease resistance genes in rice [J]. Journal of Integrative Plant Biology, 2007, 49(1): 112−119. doi: 10.1111/j.1744-7909.2006.00413.x
[5]	PROJECT I R G S. The map-based sequence of the rice genome [J]. Nature, 2005, 436(7052): 793−800. doi: 10.1038/nature03895
[6]	YOSHIDA S. Molecular regulation of leaf senescence [J]. Current Opinion in Plant Biology, 2003, 6(1): 79−84. doi: 10.1016/S1369526602000092
[7]	王备芳, 陈玉宇, 张迎信, 等. 水稻早衰突变体es5的鉴定及其突变基因的精细定位 [J]. 中国农业科学, 2018, 51(4):613−625. doi: 10.3864/j.issn.0578-1752.2018.04.002 WANG B F, CHEN Y Y, ZHANG Y X, et al. Identification and fine mapping of an early senescent leaf mutant Es5 in Oryza sativa L [J]. Scientia Agricultura Sinica, 2018, 51(4): 613−625.(in Chinese) doi: 10.3864/j.issn.0578-1752.2018.04.002
[8]	SAKURABA Y, RAHMAN M L, CHO S H, et al. The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions [J]. The Plant Journal:for Cell and Molecular Biology, 2013, 74(1): 122−133. doi: 10.1111/tpj.12110
[9]	LIN A H, WANG Y Q, TANG J Y, et al. Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice [J]. Plant Physiology, 2011, 158(1): 451−464.
[10]	LIANG C Z, WANG Y Q, ZHU Y N, et al. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice [J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(27): 10013−10018. doi: 10.1073/pnas.1321568111
[11]	KONG Z S, LI M N, YANG W Q, et al. A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice [J]. Plant Physiology, 2006, 141(4): 1376−1388. doi: 10.1104/pp.106.082941
[12]	朱永生, 蒋家焕, 蔡秋华, 等. 水稻早衰突变体w14的生理学特性分析及其基因的精细定位 [J]. 科学通报, 2021, 66(32):4144−4156. doi: 10.1360/TB-2021-0012 ZHU Y S, JIANG J H, CAI Q H, et al. Analysis of physiological characteristics of early leaf senescence mutant w14 and its gene mapping for rice [J]. Chinese Science Bulletin, 2021, 66(32): 4144−4156.(in Chinese) doi: 10.1360/TB-2021-0012
[13]	梁廷敏, 郭新睿, 陈子强, 等. 水稻材料IR65482抗稻瘟病基因鉴定与定位 [J]. 分子植物育种, 2018, 16(13):4308−4313. LIANG T M, GUO X R, CHEN Z Q, et al. Identification and mapping of a blast disease resistance gene in rice line IR65482 [J]. Molecular Plant Breeding, 2018, 16(13): 4308−4313.(in Chinese)
[14]	SUN J, YANG L M, WANG J G, et al. Identification of a cold-tolerant locus in rice (Oryza sativa L. ) using bulked segregant analysis with a next-generation sequencing strategy [J]. Rice (New York, N Y ), 2018, 11(1): 24.
[15]	SALUNKHE A S, POORNIMA R, PRINCE K S J, et al. Fine mapping QTL for drought resistance traits in rice (Oryza sativa L. ) using bulk segregant analysis [J]. Molecular Biotechnology, 2011, 49(1): 90−95. doi: 10.1007/s12033-011-9382-x
[16]	LAFARGE S, MONTANÉ M H. Characterization of Arabidopsis thaliana ortholog of the human breast cancer susceptibility gene 1: AtBRCA1, strongly induced by gamma rays [J]. Nucleic Acids Research, 2003, 31(4): 1148−1155. doi: 10.1093/nar/gkg202
[17]	BLOCK-SCHMIDT A S, DUKOWIC-SCHULZE S, WANIECK K, et al. BRCC36A is epistatic to BRCA1 in DNA crosslink repair and homologous recombination in Arabidopsis thaliana [J]. Nucleic Acids Research, 2010, 39(1): 146−154.
[18]	TRAPP O, SEELIGER K, PUCHTA H. Homologs of breast cancer genes in plants [J]. Frontiers in Plant Science, 2011, 2: 19.
[19]	EINSET J, COLLINS A R. DNA repair after X-irradiation: Lessons from plants [J]. Mutagenesis, 2014, 30(1): 45−50.