番茄<i>SlSIP1L12</i>基因负调控种子萌发的研究

崔宝禄; 陈国平

doi:10.19303/j.issn.1008-0384.2021.02.005

番茄SlSIP1L12基因负调控种子萌发的研究

doi: 10.19303/j.issn.1008-0384.2021.02.005

崔宝禄^1,,
陈国平²

1.
黔南民族师范学院，贵州　都匀　558000
2.
重庆大学，重庆　沙坪坝　400044

基金项目: 国家自然科学基金（31960605）；贵州省教育厅拔尖人才项目（黔教合[2016]111）；贵州省科技厅基础研究计划项目（[2018]1144）；贵州省植物学重点支持学科项目（qnsyzw1802）；黔南民族师范学院重大科研创新基金项目（QNSY2018BS019、QNSY2018PT001）

详细信息

作者简介:
崔宝禄（1979−），男，博士，副教授，研究方向：植物分子生物学（E-mail：cuibaolu98@163.com）

中图分类号: Q 789
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出版历程
- 收稿日期: 2020-08-04
- 修回日期: 2020-11-09
- 网络出版日期: 2021-02-08
- 刊出日期: 2021-02-28

Correlation between SlSIP1L12 Expression and Seed Germination of Tomato Plants

CUI Baolu^1
,,
CHEN Guoping²

1.
Qiannan Normal University for Nationalities, Duyun, Guizhou　558000, China
2.
Chongqing University, Shapingba, Chongqing　400044, China

摘要

摘要: 目的鉴定番茄中Trihelix转录因子SIP1亚家族SlSIP1L12基因的生物功能，方法利用RT-PCR分析了SlSIP1L12基因的表达模式、对外源激素和非生物胁迫的响应；利用RNAi干扰技术获得了SlSIP1L12转基因植株，利用ELISA鉴定转基因种子体内ABA的含量。结果（1）基因克隆分析表明：番茄AC⁺⁺中，SlSIP1L12基因编码长度为1 125bp，编码374个氨基酸；进化分析表明，番茄SlSIP1L12基因进化明显。（2）表达模式证实：SlSIP1L12基因主要在茎、成熟叶中表达，花和果实中次之。（3）外源处理显示：SlSIP1L12受外源激素ABA和脱水胁迫诱导，说明SlSIP1L12功能与ABA有关。（4）SlSIP1L12转基因株系的种子发芽速度快于对照；相同条件下，胚根伸长速度明显比对照快。（5）发芽7 d时，转基因种子体内ABA含量明显小于对照。结论 SlSIP1L12基因负调控番茄种子萌发的机制与ABA含量密切相关。
- 番茄 /
- SlSIP1L12基因 /
- 种子萌发 /
- ABA响应
Abstract: Objective Role of SlSIP1L12, an SIP1 subfamily gene of trihelix transcription factors, played in tomato seed germination was investigated. Method Expressions under and responses to phytohormone and abiotic stress of SlSIP1L12 were tested by RT-PCR. The RNAi technology was used to suppress the SlSIP1L12 expression to reveal its function on the seed germination. ELISA was applied to detect the endogenous ABA contents in the seeds. Result (1) The length of SlSIP1L12 was 1 125bp in AC⁺⁺ that encoded 374 amino acids. The phylogenetic analysis confirmed that SIP1 genes had indeed evolved in AC⁺⁺. (2) SlSIP1L12 was primarily expressed in the stems and mature leaves, and secondly, in the flowers and fruits. (3) The suppressed expression of SlSIP1L12 could be artificially induced by ABA or dehydration, which implied a close relationship between the gene and ABA. (4) The germination of the SlSIP1L12 transgenic seeds was faster and grew to longer radicles than control. (5) ABA contents were significantly reduced in the transgenic seeds 7d after germination. Conclusion The downregulated SlSIP1L12 that stimulated germination of tomato seeds was postulated to be closely associated with the ABA response of the plant.
- Solanum lycopersicom /
- SlSIP1L12 /
- seed germination /
- ABA response

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图 1 番茄果实不同发育时期

注：IMG：未成熟果实；MG：成熟果实；B：Breaker；B+4：破色后4 d；B+7：破色后7 d。

Figure 1. Photos of tomatoes at developmental stages

Note: IMG：immature green; MG：Mature green; B：Breaker; B + 4：4 days after Breaker; B + 7：7 days after Breaker.

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图 2 SIP1亚家族蛋白结构

注：完全一致的氨基酸序列采用黑色底；相似的采用灰色底。高度保守区的位置及可能结构位于序列上部；双元或单元核定位信号（NLS）采用红色方框。螺旋（Helix），中心区（Central domain）

Figure 2. The structure of SIP1 proteins

Note: Identical amino acid sequences are shaded in black, and similar ones in gray. Locations of highly conserved subdomains and possible structure are indicated on top of sequences, and degenerate bipartite or monopartite nuclear localization signal (NLS) by red box; helix; central domain.

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图 3 生物进化分析SIP1亚家族蛋白质

注：番茄（Solanum lycopersicom, Sl），油菜（Brassica napus, Bn），拟南芥（Arabidopsis thaliana, At）SlSIP1L1（Solyc01g088820）；SlSIP1L2（Solyc01g096470）；SlSIP1L3（Solyc02g076810）；SlSIP1L4（Solyc03g122030）；SlSIP1L5（Solyc05g018350）；SlSIP1L6（Solyc06g051120）；SlSIP1L7（Solyc06g083430）；SlSIP1L8（Solyc07g055100）；SlSIP1L9（Solyc08g007170）；SlSIP1L10（Solyc09g008850）；SlSIP1L11（Solyc12g010890）；SlSIP1L12*（Solyc12g043090）；ASIL1（At1g54060）；ASIL2（At3g14180）；BnSIP1-1（CDX73485）

Figure 3. Phylogenetic analysis of SIP1 proteins

Note: Si: Solanum lycopersicom; Bn: Brassica napus; At: Arabidopsis thaliana. SlSIP1L1 (Solyc01g088820); SlSIP1L2 (Solyc01g096470); SlSIP1L3 (Solyc02g076810); SlSIP1L4 (Solyc03g122030); SlSIP1L5 (Solyc05g018350); SlSIP1L6 (Solyc06g051120); SlSIP1L7 (Solyc06g083430); SlSIP1L8 (Solyc07g055100); SlSIP1L9 (Solyc08g007170); SlSIP1L10 (Solyc09g008850); SlSIP1L11 (Solyc12g010890); SlSIP1L12* (Solyc12g043090); ASIL1 (At1g54060); ASIL2 (At3g14180); BnSIP1-1 (CDX73485).

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图 4 AC⁺⁺不同组织中SlSIP1L12的表达模式

注：（1）RT：根；ST：茎；YL：幼叶；ML：成熟叶；SL：老叶；FL：花；IMG：未成熟果实；MG：成熟果实；B：破色期；B+4：破色后4天；B+7：破色后7天。（2）误差代表了样本数为3时的标准误差，采用t分布检测，字母代表P＜0.05。

Figure 4. Expressions of SlSIP1L12 in different tissues of AC⁺⁺

Note：RT: Roots; ST: Stems; YL: Young Leaves; ML: Mature Leaves; SL: Senescent Leaves; FL: Flower; IMG: Immature Green fruits; MG: Mature Green fruits; B: Break fruits; B+4: 4 d after Break fruits; B+7: 7 d after Break fruits. Error bars represent standard error of mean (n = 3). Different letters indicate significant differences between control and transgenic plants at P ＜ 0.05 by t test.

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图 5 不同处理下SlSIP1L12基因的表达模式分析

注：A：SlSIP1L12对外源激素处理的响应，Wate: 喷水; IAA: 吲哚乙酸; GA3: 赤霉素; MeJA: 茉莉酸甲酯; ABA: 脱落酸; ACC:1-氨基环丙烷羧酸；B：SlSIP1L12对非生物胁迫的响应，Water: 喷水; Dehydration: 脱水; Wounding: 伤诱导；误差代表了样本数为3时的标准误差，采用t分布检测，（*）代表P＜0.05。

Figure 5. Expressions of SlSIP1L12 under treatments by different stimuli

Note: A: response of SlSIP1L12 to hormone stimuli; B: response of SlSIP1L12 to abiotic stress. Error bars represent standard error of mean (n = 3); * at P ＜ 0.05 by t test.

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图 6 转基因不同株系中SlSIP1L12的表达水平

Figure 6. Expressions of SlSIP1L12 in RNAi-SlSIP1L12 lines

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图 7 转基因植株和AC⁺⁺种子的发芽试验

注：种子发芽势的比对（A）；种子萌发7 d时的比对（B）；种子萌发7 d时胚根长度统计分析（C）。误差代表了样本数为3时的标准误差，采用t分布检测，**代表P＜0.01，*代表P＜0.05。

Figure 7. Seed germination of transgenic and control plants

Note: Statistical analyses on germination rate (A), 7d after germination (B), and radicle length on 7^th day (C). Error bars represent standard error of mean (n = 3). Significant difference between control and transgenic plants by t test at P＜ 0.01 (**), and at P ＜ 0.05 (*).

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图 8 沉默株系内下游基因与相关激素的检测

注：误差代表了样本数为3时的标准误差，采用t分布检测，**代表 P＜0.01，*代表P＜0.05。

Figure 8. Expressions of downstream genes in transgenic and control plants

Note: Error bars represent standard error of mean (n = 3). Significant difference between control and transgenic plants by t test at P＜0.01 (**), and at P＜0.05 (*).

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表 1 引物设计

Table 1. Primers applied

引物名称 Primer names	序列（5′ → 3′） Sequences（5′ → 3′）	引物用途 Primer usage
cSlSIP1L12-F	TTGGTTGGTTTCTTGTGCTTC	基因全长片段扩增 Cloned segment of SlSIP1L12
cSlSIP1L12-R	CTCCATAATGTTATTATTGACTGTTGA	基因全长片段扩增 Cloned segment of SlSIP1L12
SlSIP1L12-F	CTTCCTGTTCACGACCCTTC	沉默片段扩增 RNAi sequence
SlSIP1L12-R	CTCTCAATTTTGGACCCCC	沉默片段扩增 RNAi sequence
SlSIP1L12-EcoRI-XbaI-F	CGGAATTCTCTAGACTTCCTGTTCACGACCCTTC	构建沉默载体 Construction of RNAi vector
SlSIP1L12-KpnI-HindIII-R	GGGGTACCAAGCTTCTCTCAATTTTGGACCCCC	构建沉默载体 Construction of RNAi vector
qSlCAC-F	CCTCCGTTGTGATGTAACTGG	基因器官中表达内参 Reference gene in tissues
qSlCAC-R	ATTGGTGGAAAGTAACATCATCG	基因器官中表达内参 Reference gene in tissues
qSlEF1α-F	TACTGGTGGTTTTGAAGCTG	处理后基因表达内参 Reference gene for treatments
qSlEF1α-R	AACTTCCTTCACGATTTCATCATA	处理后基因表达内参 Reference gene for treatments
qSlSIP1L12-F	GCATATTTTCTTGAATTAGATAAAAGGG	基因表达定量引物 Primers for RT-PCR
qSlSIP1L12-R	TAGCAACTAGATGAACAAGTTTCTCCAT	基因表达定量引物 Primers for RT-PCR

下载: 导出CSV

表 2 氨基酸序列一致性分析

Table 2. The similarity of SIP1 proteins

蛋白 Proteins	SlSIP1L12/%	蛋白 Proteins	SlSIP1L12/%	蛋白 Proteins	SlSIP1L12/%
SlSIP1L1	21	SlSIP1L6	31.8	SlSIP1L11	25.4
SlSIP1L2	20.1	SlSIP1L7	17.8	ASIL1	34.9
SlSIP1L3	30.7	SlSIP1L8	24.1	NtSIP1	24.4
SlSIP1L4	37.1	SlSIP1L9	18.9
SlSIP1L5	47.2	SlSIP1L10	17.3

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