生物技术通报 ›› 2022, Vol. 38 ›› Issue (9): 158-166.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1444
陈光1,2,3(), 李佳4, 杜瑞英1,2,3, 王旭1,2,3()
收稿日期:
2021-11-18
出版日期:
2022-09-26
发布日期:
2022-10-11
作者简介:
陈光,男,博士,副研究员,研究方向:植物营养与环境胁迫;E-mail: 基金资助:
CHEN Guang1,2,3(), LI Jia4, DU Rui-ying1,2,3, WANG Xu1,2,3()
Received:
2021-11-18
Published:
2022-09-26
Online:
2022-10-11
摘要:
通过盐胁迫筛选EMS诱变的粳稻突变体库,挖掘盐逆境响应关键基因,鉴定出一个盐敏感突变体。在100 mmol/LNaCl处理下,突变体严重萎蔫,叶片褪绿黄化,地上部Na+的积累显著高于野生型。盐胁迫后,突变体叶片的光合速率和叶绿素含量均显著低于野生型,并且源叶中的蔗糖含量增加,而根中的蔗糖只有野生型的83%。突变体源叶中参与糖转运的几个关键基因表达下调,进一步表明糖分向库器官根系的分配受抑制。突变体库组织中较低的蔗糖含量不能满足生长发育对碳营养的需求,导致水稻耐盐性降低。通过图位克隆确定候选基因,将其命名为SS2,互补实验可以恢复突变体的盐敏感表型,盐胁迫下生长的互补转基因株系与野生型植株无显著差异。综上所述,SS2通过调控体内糖分转运,对水稻的生长和盐胁迫响应起重要作用。
陈光, 李佳, 杜瑞英, 王旭. 水稻盐敏感突变体ss2的鉴定与基因功能分析[J]. 生物技术通报, 2022, 38(9): 158-166.
CHEN Guang, LI Jia, DU Rui-ying, WANG Xu. Identification and Gene Functional Analysis of Salinity-hypersensitive Mutant ss2 in Rice[J]. Biotechnology Bulletin, 2022, 38(9): 158-166.
基因名称 Gene name | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') |
---|---|---|
UBQ5 | CTCGCCGACTACAACATCCA | TCTTGGGCTTGGTGTACGTCTT |
OsSUT4 | CGCCGGCGGTGGCGGCCTCA | CGTGAGGAGCGAGAGCTGA |
OsSWEET11 | GACGTTCTTGCAGGTGTACA | TAGCGGACGATGTAGGCGGC |
OsSWEET14 | TTCCCAACGTGCTGGGCTTCT | GCACCTCGCGGGTCTTGACG |
OsMT | GCTGCCAGGCAGGAAGCT | GGTTCCAGTTTCACCACGACA |
表1 荧光定量PCR所用引物
Table 1 Primer sequences used for RT-qPCR assays
基因名称 Gene name | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') |
---|---|---|
UBQ5 | CTCGCCGACTACAACATCCA | TCTTGGGCTTGGTGTACGTCTT |
OsSUT4 | CGCCGGCGGTGGCGGCCTCA | CGTGAGGAGCGAGAGCTGA |
OsSWEET11 | GACGTTCTTGCAGGTGTACA | TAGCGGACGATGTAGGCGGC |
OsSWEET14 | TTCCCAACGTGCTGGGCTTCT | GCACCTCGCGGGTCTTGACG |
OsMT | GCTGCCAGGCAGGAAGCT | GGTTCCAGTTTCACCACGACA |
标记 Marker | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') |
---|---|---|
SS11 | GCAACTGGTGGAGTCTATTT | CATGCTAACATGAGGTGATC |
SS12 | ACGCCTCCCAAGTCGAAAGG | GGTGGGCCTCGATTGTAAGTAG |
SS10 | TTGGCTCTTCTCCTTAGTAT | CATTTGTATCTTGTGAACGT |
SS3 | CCAATGTTTGCTCCAGAT | TTCAATGACCCACGTCCC |
SS4 | TGGTTTTCCTTGTTGCTG | GCTTGCGGCTCTGCTTAC |
SS20 | TACAGGTATGCTGCTTTTCC | CTGGTCCTTTTCATTCTAAC |
SS21 | AAAACATGCTCCAACAGCCT | CCAAATGTAGCCAGTGAGGA |
SS22 | GGAGGAGTTCATTTGAGGCG | CTGGGTGGGCTAGGAAGTAA |
SS23 | ATACCTCCTTGTATTCGCACT | CGATCGATTGCCACATTATA |
SS24 | AGCGTGAATCTAATAGCACT | CGTTCAACAAGACCCAATAC |
SS25 | TTGTAACCGTCGATTTCGTTC | CCGCTCCGTCACTCTACTACC |
SS16 | CCTCCGACCTCAGCACCTGC | GTTGGCGTCCGCTGCTCCTG |
SS17 | AGGTAGGCGTGGCGATCAAC | CTTCTCCGGTCACCATCCAC |
SS27 | TTGGCGATTAATGATCCGGGAAC | CGTTCGTGCCGGTGATGTCG |
SS29 | ACAACAGTTCTTCACCAGAG | GTAGTATAAATTGTAATAGCTCAA |
SS32 | GTTAAATGAATCATCAGGAT | AGTAGTCTTGAATTCGCTGT |
表2 基因定位所用的标记引物
Table 2 Marked primer used for gene mapping
标记 Marker | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') |
---|---|---|
SS11 | GCAACTGGTGGAGTCTATTT | CATGCTAACATGAGGTGATC |
SS12 | ACGCCTCCCAAGTCGAAAGG | GGTGGGCCTCGATTGTAAGTAG |
SS10 | TTGGCTCTTCTCCTTAGTAT | CATTTGTATCTTGTGAACGT |
SS3 | CCAATGTTTGCTCCAGAT | TTCAATGACCCACGTCCC |
SS4 | TGGTTTTCCTTGTTGCTG | GCTTGCGGCTCTGCTTAC |
SS20 | TACAGGTATGCTGCTTTTCC | CTGGTCCTTTTCATTCTAAC |
SS21 | AAAACATGCTCCAACAGCCT | CCAAATGTAGCCAGTGAGGA |
SS22 | GGAGGAGTTCATTTGAGGCG | CTGGGTGGGCTAGGAAGTAA |
SS23 | ATACCTCCTTGTATTCGCACT | CGATCGATTGCCACATTATA |
SS24 | AGCGTGAATCTAATAGCACT | CGTTCAACAAGACCCAATAC |
SS25 | TTGTAACCGTCGATTTCGTTC | CCGCTCCGTCACTCTACTACC |
SS16 | CCTCCGACCTCAGCACCTGC | GTTGGCGTCCGCTGCTCCTG |
SS17 | AGGTAGGCGTGGCGATCAAC | CTTCTCCGGTCACCATCCAC |
SS27 | TTGGCGATTAATGATCCGGGAAC | CGTTCGTGCCGGTGATGTCG |
SS29 | ACAACAGTTCTTCACCAGAG | GTAGTATAAATTGTAATAGCTCAA |
SS32 | GTTAAATGAATCATCAGGAT | AGTAGTCTTGAATTCGCTGT |
图1 正常和盐胁迫条件下SS2突变对幼苗期水稻生长的影响 A:幼苗期ss2突变体及WT在正常和盐胁迫条件下的生长情况,黄色标尺=1 cm,白色标尺=5 cm;B-E:在正常和盐胁迫条件下ss2突变体及WT的株高(B),地上部鲜重(C),Na+浓度(D)和钠钾浓度比(E)。数值显示的是平均值±SE(n=5),不同的字母表示在P<0.05水平下具有显著差异,下同。DW:干重
Fig.1 Effects of SS2 mutation on the growth performance of seedlings under normal and salinity stress A:Growth performance of WT and ss2 mutants seedling under normal or NaCl treatment. Yellow bar = 1 cm. White bar = 5 cm. B-E:Shoot height(B),shoot fresh weight(C),Na+ concentration([Na])(D)and[Na]/[K]ratio in the shoots(E)of the ss2 mutants under normal and salinity stress conditions. The values are means ± SE of 5 replicates. Significant differences at P < 0.05 are indicated with different letters,the same below. DW:Dry weight
图2 分蘖期WT和ss2对盐胁迫的生理响应 A:总叶绿素含量;B:叶片净光合速率。FW:鲜重,下同
Fig. 2 Physiological responses of WT and ss2 to salinity stress during tillering stage A:Total chlorophyll content. B:Net photosynthetic rate of the leaf. FW:Fresh weight,the same below
图3 盐胁迫下WT和ss2的蔗糖转运差异分析 A:源叶中蔗糖含量;B:根中蔗糖含量;C:蔗糖外运速率
Fig. 3 Comparison of sucrose transportation between WT and ss2 in response to salinity stress A:Sucrose contents of the leaf. B:Sucrose content of the root. C:Rate of sucrose export from the leaf(SER)
图4 SS2突变对WT和ss2源叶中糖转运相关基因表达的影响 定量检测的基因有OsSUT4(A),OsSWEET11(B),OsSWEET14(C)和OsMT(D)。UBQ5基因作为内参,待测基因在正常生长的WT中的表达量设为1
Fig.4 Effects of SS2 mutation on the expressions of genes related to sugar transport in WT and ss2 leaves The genes assayed were OsSUT4(A),OsSWEET11(B),OsSWEET14(C)and OsMT(D). UBQ5 was chosen as the reference sequence. The expression of to-be-detected genes in non-stressed WT plants was set to 1
图5 SS2的图位克隆 A:SS2初步定位在第5号染色体上,并用基于BAC克隆OSJNBa0057L21序列开发的标记进行精细定位;B:SS2被精细定位到标记SS23和SS24之间55 kb的物理区域,11个开放阅读框位于该区间;C:SS2候选基因LOC_Os10g41780的基因结构,箭头指示ss2的突变位点
Fig.5 Map-based cloning of SS2 A:SS2 was primarily mapped on chromosome 5 and fine mapping with the markers developed based on the sequence of BAC clone OSJNBa0057L21. B:The SS2 locus was narrowed to a 55 kb genomic DNA region between markers SS23 and SS24. Eleven open reading frames were located in the region. C:Gene structure of the SS2 candidate LOC_Os10g41780. Arrows show mutation sites of ss2
图6 盐胁迫下WT和互补转基因株系的生长差异分析 A:幼苗期转基因互补株系及WT在正常和盐胁迫条件下的生长情况,黄色标尺=1 cm,白色标尺=5 cm。B-E:在正常和盐胁迫条件下转基因互补株系及WT的地上部生物量(B),根系生物量(C),H2O2(D)和MDA含量(E)
Fig.6 Difference analysis in the growth performance of complementary transgenic plant compared with WT in response to salinity stress A:Growth performance of WT and complementary transgenic plants under normal or 100 mmol/L NaCl treatment. Yellow bar = 1 cm. White bar = 5 cm. B-E:Shoot biomass(B),root biomass(C),H2O2(D)and MDA(E)content in the shoots of the seedlings under normal and salinity stress conditions
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