Cloning and Expression of PsWRKY33 Promoter in Nane
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摘要:
目的 探讨油柰（Prunus salicina lindley）WRKY33 启动子在不同胁迫处理下的表达模式，为进一步深入研究油柰WRKY基因在油柰生长发育或抵御各种逆境胁迫中的作用机制等提供理论依据。 方法 利用MEGA 6.06软件构建PsWRKY33的系统进化树。通过染色体步移技术克隆获得该基因启动子序列，利用PlantCARE数据库分析预测PsWRKY33 5′端上游启动子区域的顺式作用元件。利用拟南芥浸花法获得转基因植株。通过对不同胁迫处理下的各片段转基因幼苗进行组织化学染色和GUS酶活性测定。 结果 系统进化树分析表明PsWRKY蛋白与拟南芥WRKY33亲缘关系最近，故将该基因命名为PsWRKY33 。获得PsWRKY33 基因5′端上游启动子序列长度为1 872 bp，经预测分析发现该启动子区域含有ABRE、ARE、LTR、MYB和W-box等响应不同植物激素等顺式作用元件，基于此构建3个不同长度的缺失片段。对转基因植株不同组织GUS染色发现PsWRKY33 启动子主要表达在叶、花瓣和花序梗上，且随着片段的缺失其表达越低。在不同胁迫处理下，其GUS活性表达程度不同。在低温胁迫条件下，不同长度的PsWRKY33启动子片段均受到不同程度的诱导上调表达；而在SA胁迫下，各片段呈现出不同程度的诱导下调表达。 结论 PsWRKY33基因启动子 可能参与调节油柰应答低温胁迫及外源激素SA的响应。 Abstract:Objective Expression of PsWRKY33 promoter of Nane (Prunus salicina lindley) under stress was determined for a study on the mechanism of WRKY gene associated with the growth and stress-resistance of the plant. Method Unrooted phylogenetic tree representing relationships among protein sequences of PsWRKY33 was constructed using Nane and other plants through by MEGA 6.06 software. The promoter sequence was cloned by chromosome stepping technique. The cis-acting regulatory elements in the upstream promoter region at the 5′ end of PsWRKY33 were predicted according to the PlantCARE database. Transgenic plants were obtained by using the Arabidopsis immersion method. Histochemical staining and GUS enzyme activity were performed on the transgenic seedlings under different stresses. Result The phylogenetic tree analysis showed that the PsWRKY protein was most closely related to Arabidopsis WRKY33, thus the gene was named PsWRKY33. The 1 872 bp length of 5′ end upstream in PsWRKY33 was obtained, from Nane via genome walking technology. The promoter region contained motifs of hormones (ABRE and ARE) and biotic stresses (LTR, MYB, W-box, and other cis-acting elements). Then, the 3 5′-deletion fragments were constructed. The histochemical staining and GUS activity measurement showed that the full PsWRKY33 promoter fragment mainly expressed in leaves, petals, and peduncles, and its expression decreased with the loss of fragments. Under low temperature, the various fragments of PsWRKY33 promoter were upregulated, but downregulated under SA stress. Conclusion PsWRKY33 might response to low temperature and exogenous hormone SA stress in Nane. -
Key words:
- Prunus salicina /
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PsWRKY33
/ - promoter /
- histochemical staining /
- stress
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图 2 PsWRKY33
基因启动子序列 注：-60表示ATG上游60个核苷酸。红色粗体字母加框表示预测的顺式作用元件序列。pPsWRKY33、P1、P2、P3加箭头加黑色下划线表示为4个片段（−1 872、−852、−357和−271 bp）的特异性引物，箭头表示它们的方向。
Figure 2. Cis-elements of PsWRKY33 promoter
Note: -60 represented the 60 nucleotides upstream of the ATG. Bold red letters with boxes indicated the predicted sequence of cis-acting elements. pPsWRKY33, P1, P2 and P3 add arrows and black underlines represented the specific primers of four fragments (−1 872, −852, −357 and −271 bp), the arrows indicated their direction.
图 3 PsWRKY33
启动子各片段的GUS载体 注：不同启动子区域的数目表示PsWRKY33翻译起始ATG密码子上游的核苷酸位置。attR1和attR2限制性位点：将pDMC163载体中的CaMV35S启动子替换为PsWRKY33启动子片段。LB和RB：代表T-DNA的左右边缘；Hyg：潮霉素标记基因；GUSA：GUS基因编码区域。黄色的条形图代表PsWRKY33::GUS、PsWRKY33-P1::GUS、PsWRKY33-P2::GUS和PsWRKY33-P3::GUS结构的-1 872 bp、-852 bp、-357 bp和-271 bp PsWRKY33启动子片段。
Figure 3. Schematic diagram of GUS vector of individual PsWRKY33 promoter fragments
Note: The number of different promoter regions represents the nucleotide position upstream of the PsWRKY33 translation initiation ATG codon. The CaMV35S promoter in pDMC163 vector was replaced by PsWRKY33 promoter fragment by attR1 and attR2 restriction sites. LB and RB represent the left and right edges of T-DNA; Hyg, hygromycin B marker gene; GUS is the region that codes for the GUS gene. The yellow bars represent the -1 872 bp, -852 bp, -357 bp and -271 bp PsWRKY33 promoter fragments of PsWRKY33::GUS, PsWRKY33-P1::GUS, PsWRKY33-P2::GUS and PsWRKY33-P3::GUS, respectively.
图 4 PCR检测T2代转基因植株
注：A~D分别为pPsWRKY33、pPsWRKY33-P1、pPsWRKY33-P2和pPsWRKY33-P3转基因植株的PCR检测结果。1~8分为pPsWRKY33、pPsWRKY33-P1、pPsWRKY33-P2和pPsWRKY33-P3 8个转基因植株；9为质粒；10为野生型；11为DL 2 000 DNA maker。
Figure 4. Detectioned of transgenic Arabidopsis T2 plants by PCR
Note: A-D: PCR analysis on pPsWRKY33, pPsWRKY33-P1, pPsWRKY33-P2, and pPsWRKY33-P3 transgenic Arabidopsis, respectively; 1-8: 8 strains of pPsWRKY33, pPsWRKY33-P1, pPsWRKY33-P2, and pPsWRKY33-P3 transgenic Arabidopsis, respectively; 9: plasmid; 10: wild type; 11: DNA maker DL 2 000.
图 5 T3代转基因拟南芥GUS组织化学染色
注：A-C分别为叶片、花序、角果。
Figure 5. Histochemical localization of GUS activity in transgenic Arabidopsis T3 plants
Note: A-C: leaf, inflorescences, and silique pods, respectively.
图 6 不同胁迫处理下转基因拟南芥GUS组织染色
Figure 6. GUS staining of transgenic Arabidopsis under different stresses
图 7 转基因拟南芥在不同胁迫处理下的GUS相对活性
注：图中不同大、小写字母表示同一处理达极显著（P＜0.01）、显著（P＜0.05）差异。
Figure 7. GUS relative activity in transgenic Arabidopsis under different stresses
Note: Different uppercase and lowercase letters indicate highly significant (P＜0.01) and significant (P＜0.05) differences in the same treatment .
表 1 引物序列
Table 1. Sequences of primers
引物名称 Primer name 引物序列（5′-3′） Sequence of primers(5′-3′) PsWRKY33-GSPA GCGAAGTAAGAAGAGGGAGAAACGGAG PsWRKY33-GSPB TGTTGGTGCTGGGGTCTTCATCTG PsWRKY33-GSPC CGAGAAAGTGAAGGTTGGGTGTGAA pPsWRKY33-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGTTCTGCCTTGTTTGCT pPsWRKY33-P1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGATCGAATTTTTTTTTCATA pPsWRKY33-P2-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATTATATTGGTTTACATACA pPsWRKY33-P3-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAACCCACGAGCTTTGACCA pPsWRKY33-R GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAGGGGGGGAGAGAGAGAG 
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