植物学报 ›› 2019, Vol. 54 ›› Issue (1): 46-57.DOI: 10.11983/CBB18045
收稿日期:
2018-02-12
接受日期:
2018-05-21
出版日期:
2019-01-01
发布日期:
2019-07-31
通讯作者:
徐凡
基金资助:
Shengnan Cui1,2,Yihan Zhang1,Fan Xu1,*()
Received:
2018-02-12
Accepted:
2018-05-21
Online:
2019-01-01
Published:
2019-07-31
Contact:
Fan Xu
摘要: 蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在ProOsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。
崔胜男,张艺函,徐凡. 异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老. 植物学报, 2019, 54(1): 46-57.
Shengnan Cui,Yihan Zhang,Fan Xu. Heterologous Overexpression of Rice OsSAPP3 Gene Promotes Leaf Senescence in Transgenic Arabidopsis. Chinese Bulletin of Botany, 2019, 54(1): 46-57.
Primer name | Primer sequence (5'-3') | Annotation |
---|---|---|
CDSOsSAPP3-F | CCCGGGATGCTATCTGCTGCGATGGAATACTT | PCR |
CDSOsSAPP3-R | GAACTCTATGTAGGAACTGAGAATGGAGCAAGG | PCR |
cGVG-SAPP3-F | GTCGACATGCTATCTGCTGCGATGGAATAC | PCR |
cGVG-SAPP3-R | ATCGATCTATGTAGGAACTGAGAATGGAGCAA | PCR |
ProOsSAPP3-F | AAGCTTTCATATCCGTTGTTCCGTAGCG | PCR |
ProOsSAPP3-R | TCTAGACCTTCATCTCCGACACCTCCC | PCR |
表1 引物序列
Table 1 Sequences of primers
Primer name | Primer sequence (5'-3') | Annotation |
---|---|---|
CDSOsSAPP3-F | CCCGGGATGCTATCTGCTGCGATGGAATACTT | PCR |
CDSOsSAPP3-R | GAACTCTATGTAGGAACTGAGAATGGAGCAAGG | PCR |
cGVG-SAPP3-F | GTCGACATGCTATCTGCTGCGATGGAATAC | PCR |
cGVG-SAPP3-R | ATCGATCTATGTAGGAACTGAGAATGGAGCAA | PCR |
ProOsSAPP3-F | AAGCTTTCATATCCGTTGTTCCGTAGCG | PCR |
ProOsSAPP3-R | TCTAGACCTTCATCTCCGACACCTCCC | PCR |
图1 ProOsSAPP3-GUS转基因拟南芥不同发育时期GUS组织化学染色 (A) 染色前的转基因拟南芥, 从上到下, 从左到右, 依次为Stage 0.5、Stage 1.02、Stage 1.04、Stage 1.10、Stage 6.0、Stage 6.9; (B) 染色后的转基因拟南芥, 从上到下, 从左到右苗龄同染色前; (C) 经MOCK溶液(激素溶剂)处理的转基因拟南芥; (D) 经10 μmol?L-1 ABA溶液处理的转基因拟南芥; (E) 经10 μmol?L-1 6-BA溶液处理的转基因拟南芥。Bars=5 mm
Figure 1 GUS histochemical staining of ProOsSAPP3-GUS transgenic Arabidopsis thaliana at different developmental stages (A) From top to bottom, from left to right, the transgenic Arabidopsis thaliana before stain: Stage 0.5, Stage 1.02, Stage 1.04, Stage 1.10, Stage 6.0, Stage 6.9; (B) From top to bottom, from left to right, the transgenic Arabidopsis thaliana was stained in turn, and the seedling age was identical to that before staining; (C) Transgenic Arabidopsis thaliana treated with MOCK solution (hormone solvent); (D) Transgenic Arabidopsis thaliana treated with 10 μmol?L-1 ABA solution; (E) Transgenic Arabidopsis thaliana treated with 10 μmol?L-1 6-BA solution. Bars=5 mm
图2 组成型异源过表达OsSAPP3基因导致转基因拟南芥生长发育异常 (A) 35S-OsSAPP3转基因与野生型拟南芥的基因表达水平; (B) WT为野生型拟南芥植株; 35S-OsSAPP3为35S-OsSAPP3转基因拟南芥株系; 图左上为营养土中生长14天的幼苗, 图左下为营养土中生长21天的植株, 图右为营养土中培养75天的植株。图中数据为平均值±标准差, n=10, 实验经3次生物学重复。** P<0.01。Bars=1 cm
Figure 2 Heterologous expression of OsSAPP3 gene lead to abnormal growth and development of transgenic Arabidopsis (A) 35S-OsSAPP3 transgenic Arabidopsis and wild-type Arabidopsis gene expression levels; (B) Wild-type Arabidopsis plant (WT) and 35S-OsSAPP3 transgenic Arabidopsis line (35S-OsSAPP3). The top left panel was the 14-day-old seedling growing in the nutrient soil, the bottom left is the 21-day-old plant growing in the nutrient soil, and the right panel was the plants cultured in nutrient soil for 25 days. Values are means±SD of one representative biological replicate (n=10) out of three, ** P<0.01. Bars=1 cm
图3 外源诱导OsSAPP3异源过表达导致转基因拟南芥无法正常萌发 (A)-(C) DEX诱导36小时, 各转基因种子与对照种子萌发情况; (D)-(F) DEX诱导48小时, 各转基因种子与对照种子萌发情况; (G)-(I) DEX诱导72小时, 各转基因种子与对照种子萌发情况; (J) 外源诱导不同时长, OsSAPP3基因的表达变化情况; (K) 外源诱导不同时长, WRKY6基因的表达变化情况; (L) GVG-OsSAPP3转基因拟南芥纯合株系的基因表达水平。数据为平均值±标准差, n=10, 实验经3次生物学重复。* P<0.05, ** P<0.01。Bars=1 cm
Figure 3 Exogenously induced OsSAPP3 heterologous over expression resulted in the inability of germination of transgenic Arabidopsis (A)-(C) DEX induced 36 h, the germination of transgenic and control seeds; (D)-(F) DEX induced 48 h, the germination of transgenic and control seeds; (G)-(I) DEX induced 72 h, the germination of transgenic and control seeds; (J) The expression of OsSAPP3 induced by exogenous factors at different times; (K) Exogenous induction of WRKY6 expression changes at different times; (L) Gene expression levels of GVG-OsSAPP3 transgenic Arabidopsis homozygous lines. Values are means±SD of one representative biological replicate (n=10) out of three. * P<0.05, ** P<0.01. Bars=1 cm
Number of rosette leavesa | 5% significant level | 1% very significant level | Rosette leaf sizeb | 5% significant level | 1% very significant level | |
---|---|---|---|---|---|---|
CK | 9.4 | c | A | 2.84 | d | C |
Line2 | 10.2 | bc | A | 2.3 | ab | BC |
Line20 | 11.8 | abc | A | 1.9 | bc | AB |
Line14 | 12.6 | ab | A | 1.5 | cd | A |
Table 2 Phenotypic data of Arabidopsis GVG-OsSAPP3 transgenic lines and control (CK)
Number of rosette leavesa | 5% significant level | 1% very significant level | Rosette leaf sizeb | 5% significant level | 1% very significant level | |
---|---|---|---|---|---|---|
CK | 9.4 | c | A | 2.84 | d | C |
Line2 | 10.2 | bc | A | 2.3 | ab | BC |
Line20 | 11.8 | abc | A | 1.9 | bc | AB |
Line14 | 12.6 | ab | A | 1.5 | cd | A |
图4 外源诱导OsSAPP3基因异源过表达促进转基因拟南芥成熟苗提前衰老 (A) 喷施处理5天后的转化对照(CK)和GVG-OsSAPP3转基因拟南芥; (B) 苗龄24天的CK和GVG-OsSAPP3转基因拟南芥的代表植株; (C) CK和GVG-OsSAPP3转基因拟南芥的抽薹进程; (D) 苗龄34天的CK和GVG-OsSAPP3转基因拟南芥植株; (E) CK和GVG-OsSAPP3转基因拟南芥的开花进程; (F) 喷施处理后21天的CK和GVG-OsSAPP3转基因拟南芥; (G) 苗龄26天的CK和GVG-OsSAPP3转基因拟南芥第4、5和6片叶的叶绿素含量。数据为平均值±标准差, n=10, 实验经3次生物学重复。Bars=1 cm
Figure 4 Exogenous-induced OsSAPP3 heterologous over expression promotes premature aging of transgenic Arabidopsis mature seedlings (A) Arabidopsis control (CK) and GVG-OsSAPP3 transgenic Arabidopsis after spray treatment for 5 days; (B) 24-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis plants; (C) The CK and GVG-OsSAPP3 transgenic Arabidopsis bolting process; (D) 34-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis plants; (E) The CK and GVG-OsSAPP3 transgenic Arabidopsis flowering process; (F) CK and GVG-OsSAPP3 transgenic Arabidopsis after spray treatment for 21 days; (G) The chlorophyll contents of the fourth, fifth and sixth leaf of 26-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis. Values are means±SD of one representative biological replicate (n=10) out of three. Bars=1 cm
图5 30 μmol?L-1 DEX与MOCK溶液处理24小时后, 转化对照(CK)和GVG-OsSAPP3转基因拟南芥第5和6片叶的衰老标志基因表达变化
Figure 5 Changes of senescence marker gene expression in the fifth and sixth leaves of control (CK) and GVG-OsSAPP3 transgenic Arabidopsis after 30 μmol?L-1 DEX and MOCK solution treatment for 24 hoursValues are means±SD of one representative biological replicate (n=10) out of three. * P<0.05, ** P<0.01.
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