中国农业科学 ›› 2021, Vol. 54 ›› Issue (12): 2487-2498.doi: 10.3864/j.issn.0578-1752.2021.12.001
收稿日期:
2020-11-18
接受日期:
2021-01-15
出版日期:
2021-06-16
发布日期:
2021-06-24
通讯作者:
王平荣
作者简介:
张翔宇,E-mail:基金资助:
ZHANG XiangYu(),GUO Jia,WANG San,CHEN CongPing,SUN ChangHui,DENG XiaoJian,WANG PingRong()
Received:
2020-11-18
Accepted:
2021-01-15
Online:
2021-06-16
Published:
2021-06-24
Contact:
PingRong WANG
摘要:
【目的】对水稻粒宽突变体gw87(grain width 87)进行表型鉴定、遗传分析、基因定位及候选基因分析,为探明该基因调控水稻籽粒大小的分子机制及应用潜力奠定基础。【方法】利用甲基磺酸乙酯诱变籼稻恢复系材料676R,获得一份籽粒宽度和千粒重显著增加的突变体gw87。对该突变体进行表型观察、农艺性状调查及外源油菜素内酯(BL)敏感性、叶绿素含量、光合参数测定,了解其表型特征及生理特性。调查gw87与676R杂交F1的表型和F2群体的分离情况,分析其遗传行为;选取该群体中的突变植株进行高通量测序,并利用gw87与粳稻品种日本晴杂交的F2代作为定位群体,通过MutMap分析和分子标记定位,遴选候选基因并进行DNA和cDNA测序验证。利用qRT-PCR分析gw87和676R中BR合成途径基因OsDWARF4、D11和D2的表达差异。【结果】与野生型亲本676R相比,gw87突变体的株高降低,节间缩短,其中,倒一节间长度缩短最大,并呈现出扭曲的形态;叶片长度减少,宽度增加;单株有效穗数、主穗穗长和结实率显著降低,但籽粒宽度和千粒重显著增大。BL敏感性试验显示,gw87突变体幼苗对外源BL的敏感性降低。光合色素和光合参数测定表明,gw87光合色素含量增加,光合速率也有所增加。遗传分析表明gw87的突变性状是由1对隐性核基因控制。MutMap分析显示gw87突变基因位于第5染色体中部,在该染色体区域仅有1个碱基突变引起编码氨基酸变化;分子标记连锁分析表明该突变基因位于InDel标记X2和X3之间约101 kb的染色体区域;综合这两方面分析结果,最后遴选出gw87候选基因是编码一个含有AP2/EREBP DNA绑定结构域的转录因子基因LOC_Os05g32270。对该候选基因进行DNA和cDNA测序验证,发现gw87突变体中该基因DNA的第1 041位的碱基由G突变为A,导致与该位点相邻的76 bp内含子序列被剪接为外显子,引起阅读框移码,蛋白翻译提前终止。qRT-PCR分析显示gw87突变体中BR合成途径基因的表达量显著上调,表明gw87突变体中BR信号减弱。【结论】gw87是smos1、shb、rla1、ngr5的新等位突变体,但与这些突变体表型不同,gw87籽粒的宽度和千粒重显著增加,可能是LOC_Os05g32270的突变位点不同,导致其编码蛋白的功能活性不同所造成。
张翔宇,郭佳,王三,陈聪萍,孙昌辉,邓晓建,王平荣. 水稻粒宽突变体gw87的基因定位及候选基因分析[J]. 中国农业科学, 2021, 54(12): 2487-2498.
ZHANG XiangYu,GUO Jia,WANG San,CHEN CongPing,SUN ChangHui,DENG XiaoJian,WANG PingRong. Gene Mapping and Candidate Gene Analysis of Grain Width Mutant gw87 in Rice[J]. Scientia Agricultura Sinica, 2021, 54(12): 2487-2498.
表2
gw87与其野生型亲本676R主要农艺性状的比较"
性状Trait | 676R (WT) | gw87 | 比WT增减 Compared with CK (%) |
---|---|---|---|
株高 Plant height (cm) | 118.0±1.9 | 80.2±1.7 | -32.0** |
单株有效穗 No. of productive panicles per plant | 5.8±0.4 | 4.9±0.2 | -15.5* |
主穗穗长 Length of main panicle (cm) | 27.5±1.0 | 18.6±1.2 | -32.4** |
主穗一次枝梗数 No. of primary branches per main panicle | 14.8±0.5 | 16.7±1.0 | 12.8* |
主穗二次枝梗数 No. of secondary branches per main panicle | 42.5±1.1 | 30.5±1.0 | -28.2** |
主穗着粒数 Total No. of grains per main panicle | 214.6±2.5 | 216.8±5.1 | 1.0 |
结实率Seed setting rate (%) | 83.9±0.4 | 67.3±2.0 | -16.6** |
千粒重1000-grain weight (g) | 37.4±0.2 | 41.9±0.2 | 12.0** |
表4
突变体gw87与野生型676R叶片的光合参数测定"
材料 Material | 净光合速率Pn Net photosynthetic rate (μmol·m-2.s-1) | 蒸腾速率Tr Transpiration rate (μmol·m-2.s-1) | 气孔导度Gs Stomatal conductance to water vapor (μmol·m-2.s-1) | 胞间CO2浓度Ci Intercellular CO2 concentration (μmol·m-2.s-1) |
---|---|---|---|---|
676R(WT) | 21.65±2.91 | 4.52±0.36 | 0.29±0.04 | 225.04±29.92 |
gw87 | 23.16±2.32 | 4.84±0.22 | 0.26±0.02 | 198.56±26.79 |
与WT相比Compared with WT (%) | 7.0 | 7.1 | -10.3 | -11.8 |
表6
gw87突变基因的MutMap分析结果以及用于精细定位的InDel和SSR标记"
标记名称 SNP and marker name | 物理位置 Physical location (bp) | SNP指数 SNP Index | 注释基因及碱基突变位点,或分子标记引物序列 Annotated gene and its nucleotide mutation site, or marker primers |
---|---|---|---|
SNP01 | 9834287 | 0.956 | LOC_Os05g17170 downstream, G2464A |
SNP02 | 10490240 | 0.920 | LOC_Os05g18240 downstream, G1713A |
SNP03 | 11888797 | 0.909 | LOC_Os05g20290 upstream, -2662G>A |
SNP04 | 12878107 | 1 | LOC_Os05g22680 downstream,79G>A |
SNP05 | 13399064 | 0.962 | Intergenic_region, LOC_Os05g23430-LOC_Os05g23440,13399064C>T |
SNP06 | 13926842 | 0.960 | LOC_Os05g24140 upstream,-1434C>T |
SNP07 | 13966225 | 0.958 | LOC_Os05g24190 upstream,-3398C>T |
SNP08 | 14365938 | 0.906 | LOC_Os05g24790 upstream,-2425C>T |
SNP09 | 14647328 | 0.913 | LOC_Os05g25240,synonymous,93C>T|p.Cys31Cys |
SNP10 | 14908086 | 0.960 | LOC_Os05g25640 upstream_gene, -3616G>A |
X1 | 18307365 | F: TATAGCCAGGATGAGAAG, R: CTGCTGTGGATCATCTTG | |
X2 | 18753900 | F: GTACCCATCATAGCAAGA, R: AATAGGGTTGGAAGAATG | |
SNP11 | 18813290 | 1 | LOC_Os05g32270, splice_donor_variant&intron_variant,1040+1G>A |
X3 | 18854840 | F: CTATTTGCTATCGGACAC, R: ATTACTCTTTGGGTTTGG | |
SNP12 | 18932427 | 1 | LOC_Os05g32430 downstream,117G>A |
X4 | 18932825 | F: CGGCAACAAGAGGAGGAT, R: ACAGCAATGTCCGCAACGGTTTACA | |
RM163 | 19251955 | F: ATCCATGTGCGCCTTTATGAGGA, R: CGCTACCTCCTTCACTTACTAGT |
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