摘要
【目的】在京秀(Vitis vinifera‘Jingxiu’)葡萄中克隆类钙调磷酸酶B亚基蛋白基因Vv CBL4及其启动子,分析Vv CBL4的表达特性与逆境胁迫的关系。【方法】用RACE技术克隆Vv CBL4的全长,实时荧光定量PCR检测Vv CBL4在不同逆境下的表达;用同源克隆技术获得Vv CBL4的启动子,在烟草叶片中瞬时表达分析Vv CBL4启动子的活性。【结果】Vv CBL4全长为959 bp,ORF为642 bp,编码213个氨基酸,具有3个EF手钙结合结构域。Vv CBL4在根部表达量最高,其次是果实。Vv CBL4的转录本能对干旱、低温和盐胁迫处理快速做出响应。Vv CBL4启动子富含与逆境响应相关的顺式作用元件。干旱、低温和盐胁迫处理能够增强启动子活性。【结论】获得Vv CBL4及其启动子序列,Vv CBL4的表达能对逆境胁迫做出响应,Vv CBL4启动子的活性受逆境胁迫诱导,说明Vv CBL4在葡萄逆境胁迫反应中具有重要的作用。
【Objective】Grapevines are frequently challenged by various stresses during cultivation, suchas drought, high salt levels and low temperatures. In the long-term cultivation and evolution process, theyhave evolved adaptive mechanisms that allow them to survive during such periods of stress. Calcium, as asecond messenger, plays an important role in various signal transduction pathways, especially in plantstress responses. Calcineurin B-like protein-interacting protein kinases（CIPKs） belong to a Ca2＋mediatedCBL-CIPK network which provides a response to stress. In our previous study, we found that Vv CIPK10 transcripts quickly respond to drought, salt levels and low temperature. A yeast two hybrid assay showedthat Vv CIPK10 interacted with Vv CBL4. Vv CBL4 showed high homolog with Arabidopsis SOS4. Previousresearch showed that SOS4 involved during salt and drought stress. These findings prompted us to investi-gate the function of Vv CBL4 in grapevine stress responses.【Methods】We used modified SDS/phenol meth-ods to isolate the total RNA in grapevine leaves. Purified RNA was treated with DNase I to remove the ge-nomic DNA. First strand c DNA was prepared using a SMART？ RACE c DNA Amplification Kit（Takara,Dalian, China） and thereby provided primers. Based on the sequence data, gene-specific primers were de-signed, and 5＇- and 3＇-c DNA fragments were produced using an Advantage 2#174; PCR Kit. PCR productswere gel purified using a Universal DNA Purification Kit（TIANGEN, Beijing, China）, and were thencloned into p MD-19-T vectors and both strands were sequenced at the Beijing Genomics Institute. Sequences were edited and assembled into contigs using Seqman. Sequences were analyzed through the NC-BI and Ex PASy websites. Grapevine plants were treated through drought, high salt level and low tempera-ture conditions. Real-time RT-PCR（q RT-PCR） was performed using a Bio-Rad IQ5 Real-Time PCR De-tection System（Bio-Rad Laboratories, Hercules, CA, USA）. First-strand c DNA was synthesized using thePrime Script 1st strand c DNA Synthesis Kit（Ta Ka Ra, Dalian, China）. Genomic DNA was removed by DN-ase I. q RT-PCR amplification performed in a total volume of 25 μL for 45 cycles of 10 s at 95 ℃, 30 s at58 ℃, and 30 s at 72 ℃. Based on the grapevine genome sequences, a design primer to clone the promotersequence of the Vv CBL4. Cis-element of the Vv CBL4 promoter was analyzed through the Plant CARE andPLACE websites. The Vv CBL4 promoter sequence was ligated into the corresponding site of the vectorp C0380 GUS, immediately upstream of the ATG start codon for β-glucuronidase. A recombination con-struct, verified by sequencing, was introduced into the Agrobacterium tumefaciens strain GV3101 by elec-troporation. After using a Agrobacterium-mediated transient, the transformed materials and the controlwere treated through applyhing drought, high salt levels and low temperature conditions, and then detect-ing the GUS activity by using a Hitachi 850 Fluorescence spectrophotometer（Hitachi, Tokyo, Japan）.【Re-sults】The full-length of the Vv CBL4 is 959 bp was observed using an open reading frame of 642 bp with a109 bp 5＇untranslated sequence and a 208 bp 3＇untranslated region. The Vv CBL4 encodes a protein of213 aa with a calculated molecular weight of 24.5 k Da and an isoelectric point of 4.72. The deduced aminoacid sequence of the Vv CBL4 contains a 3 EF-hand calcium-binding motif. Using a phylogenetic tree anal-ysis showed that they were classified into four subfamilies. Real-time PCR results showed that Vv CBL4 presented the highest expression level in the root, a higher expression level in the fruit, and lower expres-sion levels in the tendril, inflorescence, stem and leaf. Vv CBL4 transcripts showed the same expressionmodel after drought and salt treatment. Vv CBL4 transcripts induced and reached the peak at 2 h, and thenbecame barely detectable at 6 h, reached the second peak at 12 h, and remained at a relatively low level at24 h. After cold treatment, Vv CBL4 transcripts were induced at 2 h and reached the peak at 6 h. The Vv CBL4 promoter was isolated by using the homology cloning method. The full length of the Vv CBL4 pro-moter is 1 923 bp, and the cis-element prediction showed that it contained the essential components of theeukaryotic promoter, such as TATA-box and CAAT-box. Among these cis-elements, which envolved dur-ing the stress response, were include Box-W1, CGTCA-motif, MBS, TC-rich repeats, TCA-element andW box. The Vv CBL4 promoter sequence fusion with GUS and transient expression in grapevine leaves, andthe GUS activity results showed that the negative control exhibited low GUS enzyme activity, whereas the Vv CBL4 promoter controlled GUS showed a higher level GUS activity than the GUS activity of the negativecontrol, but lower than the 35 S promoter. After drought, salt and cold treatment, the Vv CBL4 promoter con-trolled GUS showed a higher level of GUS activity than the untreated.【Conclusion】In this study, we isolat-ed and characterized Vv CBL4 and its promoter in grapevines. We demonstrated that Vv CBL4 presented thehighest expression level in the root, and showed a higher level in the fruit. The transcripts of Vv CBL4 quick-ly responed to drought, low temperatures and salt stresses. The Vv CBL4 promoter enriches the cis-elementthat is involved in stress responses. Drought, low temperatures and salt stress can enhance the promoter ac-tivities. These results indicated that Vv CBL4 plays an important role in grapevine stress responses.
出处
《果树学报》
CAS
CSCD
北大核心
2016年第4期385-392,共8页
Journal of Fruit Science
基金
河南省教育厅科学技术研究重点项目(14A210018)
河南省教育厅科技攻关项目(17020005)
河南科技大学博士科研基金(09001765)
河南科技大学创新团队计划(2015TTD003)
