扁桃AcCBF1基因VIGS载体构建与功能分析
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摘要
【目的】利用VIGS(TRV-mediated Virus Induced Gene Silencing)方法沉默扁桃(Amygdalus communis L.)AcCBF1基因表达,并初步探讨AcCBF1对低温条件下花药发育的调控。【方法】从‘纸皮’扁桃花药中克隆AcCBF1基因(729 bp),并设计4个不同长度(729、345、464、270 bp)的AcCBF1基因片段,连接到pTRV2载体上,构建VIGS载体,转化根瘤农杆菌GV3101,并用注射法侵染到扁桃花蕾内。对侵染的花器官低温处理,用半定量PCR和荧光定量PCR分析AcCBF1基因表达,以及对花器官表型进行初步分析。【结果】成功构建了3个AcCBF1基因沉默表达载体,pTRV2-AcCBF12和pTRV2-AcCBF14能显著沉默扁桃花药中AcCBF1的表达;低温处理后这两组处理的扁桃花瓣小且颜色浅,花芽和花药鲜重轻且小,与对照相比差异显著。【结论】AcCBF1在调控低温条件下对花器官表型指标发挥重要作用。该研究结果可为扁桃生殖期抗冻相关基因的功能验证和分子调控机制研究提供重要的参考和技术支持。
【Objective】To clone some fragments of the almond AcCBF1 gene and construct the gene silencing vector pTRV2-AcCBF1 mediated by tobacco rattle virus, preliminary study was carried out on the AcCBF1 regulation of flower organ development under low temperature conditions, so as to lay a theoretical foundation for the breeding of hardy-resistant varieties and further study on gene function verification.【Methods】The target gene fragment was cloned from the anther of‘Zhipi'almond by RTPCR. After the constructed expression vector was transfected into Agrobacterium GV3101, the anthers were infected by means of injection and vacuum infection. The empty vector control and infested shoots were placed in an artificial climate chamber with 70% relative humidity for hydroponics until the budswell stage(16 h during the day, 23 ℃, 8 000 lx; 8 h at night, 15 ℃, 0 lx). After the end of the night mode, the artificial climate box was adjusted to 10 ℃, 0 lx, 12 h. Then, the anthers of 30 branches of each treatment group were stripped and stored with liquid nitrogen. The remaining branches were transfered to the gradient refrigerator at 4 ℃ · h-1 to 0 ℃,0 ℃ 3 h and-2 ℃ 2 h. The remaining shoots were warmed at 4 ℃ · h-1, and the culture was continued to observe the phenotypic changes of the branches.Semi-quantitative and fluorescence quantitative analysis were performed with RNA using RT-PCR and q RT-PCR, using PdActin1 as an internal reference gene. The silencing effect of the VIGS system on the AcCBF1 gene was evaluated by phenotypic observation, semi-quantitative and fluorescence quantitative detection.【Results】The results showed that:(1) The expression level of AcCBF1 after low temperature treatment was significantly higher than that before treatment, and there was a significant difference between each treatment and the no-load control after low temperature treatment. The expression of AcCBF1 gene in almond anther was analyzed by real-time PCR. The results showed that the expression of AcCBF1 gene was significantly up-regulated in anther after low temperature treatment, reaching 2.6 times more than that before treatment. This indicated that AcCBF1 may also played a role in regulating the cold stress response to almond flower branches;(2) Four AcCBF1 gene fragments with different sizes were cloned and sequenced to construct subsequent vectors. The final sequencing result of the insert of 750 bp, 350 bp and 250 bp was consistent with the published gene sequence and can be used to construct a viral vector. The 450 bp sequence of the insert had only 30% homology with the original sequence, so that this fragment was not selected for subsequent vector construction;(3) The Agrobacterium containing pTRV1:pTRV2 and pTRV1:pTRV2-AcCBF11-4 was mixed in equal amounts, and the amygdala was infested by vacuum infestation and injection. The results showed that the flower buds were easily detached, and the subsequent experiments could not be continued. In the subsequent experiments the injection method was used to infect the flower branches;(4) Verifying gene silencing efficiency was conducted by semi-quantitative PCR and real-time PCR. The silencing of AcCBF1 by pTRV2-AcCBF12 and pTRV2-AcCBF14 was better, and the expression level of the gene decreased by about 30% compared with the control. Infecting flower branches by injection can effectively silence the expression of AcCBF1 gene, and also prove that the Amygdalus VIGS system was successfully constructed.(5) After low-temperature treatment of VIGS-treated almonds, the infecting pTRV2-AcCBF12,4 could be found in the empty vector control petals that were less open and the petals were smaller. Further collection of various tissues for quality determination revealed that the quality of the flower organs decreased after the AcCBF1 gene was silenced. The shape of the petals and anthers was also evaluated. It was found that the decrease of the AcCBF1 gene caused the longitudinal diameter and diameter of the petals to decrease significantly compared with the control; the longitudinal diameter, transverse diameter and lateral diameter of the anthers were also reduced.【Conclusion】In this study, the VIGS silencing system based on tobacco fragile virus(TRV) was established on the almond flower organ for the first time. The amygdalin carrying pTRV2-AcCBF1 was successfully used to infect the almond flower organ and the silencing effect appeared. The experiment initially proved that AcCBF1 played an important role in flower organ development due to the regulation of low temperature. The results can not only provide technical support for the identification of the downstream regulatory genes of AcCBF1 in almonds, but also lay a theoretical foundation for the discovery of the mechanism of AcCBF1 gene in the development of almond flower organs.
【Objective】To clone some fragments of the almond AcCBF1 gene and construct the gene silencing vector pTRV2-AcCBF1 mediated by tobacco rattle virus, preliminary study was carried out on the AcCBF1 regulation of flower organ development under low temperature conditions, so as to lay a theoretical foundation for the breeding of hardy-resistant varieties and further study on gene function verification.【Methods】The target gene fragment was cloned from the anther of‘Zhipi'almond by RTPCR. After the constructed expression vector was transfected into Agrobacterium GV3101, the anthers were infected by means of injection and vacuum infection. The empty vector control and infested shoots were placed in an artificial climate chamber with 70% relative humidity for hydroponics until the budswell stage(16 h during the day, 23 ℃, 8 000 lx; 8 h at night, 15 ℃, 0 lx). After the end of the night mode, the artificial climate box was adjusted to 10 ℃, 0 lx, 12 h. Then, the anthers of 30 branches of each treatment group were stripped and stored with liquid nitrogen. The remaining branches were transfered to the gradient refrigerator at 4 ℃ · h-1 to 0 ℃,0 ℃ 3 h and-2 ℃ 2 h. The remaining shoots were warmed at 4 ℃ · h-1, and the culture was continued to observe the phenotypic changes of the branches.Semi-quantitative and fluorescence quantitative analysis were performed with RNA using RT-PCR and q RT-PCR, using PdActin1 as an internal reference gene. The silencing effect of the VIGS system on the AcCBF1 gene was evaluated by phenotypic observation, semi-quantitative and fluorescence quantitative detection.【Results】The results showed that:(1) The expression level of AcCBF1 after low temperature treatment was significantly higher than that before treatment, and there was a significant difference between each treatment and the no-load control after low temperature treatment. The expression of AcCBF1 gene in almond anther was analyzed by real-time PCR. The results showed that the expression of AcCBF1 gene was significantly up-regulated in anther after low temperature treatment, reaching 2.6 times more than that before treatment. This indicated that AcCBF1 may also played a role in regulating the cold stress response to almond flower branches;(2) Four AcCBF1 gene fragments with different sizes were cloned and sequenced to construct subsequent vectors. The final sequencing result of the insert of 750 bp, 350 bp and 250 bp was consistent with the published gene sequence and can be used to construct a viral vector. The 450 bp sequence of the insert had only 30% homology with the original sequence, so that this fragment was not selected for subsequent vector construction;(3) The Agrobacterium containing pTRV1:pTRV2 and pTRV1:pTRV2-AcCBF11-4 was mixed in equal amounts, and the amygdala was infested by vacuum infestation and injection. The results showed that the flower buds were easily detached, and the subsequent experiments could not be continued. In the subsequent experiments the injection method was used to infect the flower branches;(4) Verifying gene silencing efficiency was conducted by semi-quantitative PCR and real-time PCR. The silencing of AcCBF1 by pTRV2-AcCBF12 and pTRV2-AcCBF14 was better, and the expression level of the gene decreased by about 30% compared with the control. Infecting flower branches by injection can effectively silence the expression of AcCBF1 gene, and also prove that the Amygdalus VIGS system was successfully constructed.(5) After low-temperature treatment of VIGS-treated almonds, the infecting pTRV2-AcCBF12,4 could be found in the empty vector control petals that were less open and the petals were smaller. Further collection of various tissues for quality determination revealed that the quality of the flower organs decreased after the AcCBF1 gene was silenced. The shape of the petals and anthers was also evaluated. It was found that the decrease of the AcCBF1 gene caused the longitudinal diameter and diameter of the petals to decrease significantly compared with the control; the longitudinal diameter, transverse diameter and lateral diameter of the anthers were also reduced.【Conclusion】In this study, the VIGS silencing system based on tobacco fragile virus(TRV) was established on the almond flower organ for the first time. The amygdalin carrying pTRV2-AcCBF1 was successfully used to infect the almond flower organ and the silencing effect appeared. The experiment initially proved that AcCBF1 played an important role in flower organ development due to the regulation of low temperature. The results can not only provide technical support for the identification of the downstream regulatory genes of AcCBF1 in almonds, but also lay a theoretical foundation for the discovery of the mechanism of AcCBF1 gene in the development of almond flower organs.
引文
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[2]李鹏.扁桃花蕾抗寒性分析和花药开裂过程观察[D].乌鲁木齐:新疆农业大学,2015.LI Peng. Analysis on cold resistance of almond buds and observation of the dehiscence process of almond anthers[D]. Urumqi:Xinjiang Agricultural University,2015.
[3]李鹏,田嘉,许娟,罗淑萍,李疆.鹰咀扁桃花蕾在低温应激反应中糖含量的变化[J].经济林研究,2016,34(2):62-66.LI Peng,TIAN Jia,XU Juan,LUO Shuping,LI Jiang. Changes of sugar content in flower buds of Yingzui almond in low temperature stress reaction[J]. Nonwood Forest Research,2016,34(2):62-66.
[4]钟海霞,陆婷,刘立强,李疆,李古权.不同低温胁迫下野扁桃与栽培扁桃花原基解剖结构观察[J].西北农业学报,2013,22(12):112-118.ZHONG Haixia,LU Ting,LIU Liqiang,LI Jiang,LI Guquan.Observation of the anatomical structure of floral primordium of amygdalus ledebouriana schleche and A.communis L.cv.in different low temperature stress[J]. Acta Agriculturae Boreali-occidentalis Sinica,2013,22(12):112-118.
[5]李鹏,罗淑萍,田嘉,许娟,李疆.低温冻害对扁桃花蕾抗寒机制的影响[J].经济林研究,2015,33(2):20-25.LI Peng,LUO Shuping,TIAN Jia,XU Juan,LI Jiang. Effects of frozen injury on freezing-tolerance mechanism of flower buds in almond[J]. Nonwood Forest Research,2015,33(2):20-25.
[6]李鹏,田嘉,唐开文,罗淑萍,李疆.基于隶属函数评估法的扁桃花蕾抗寒性研究[J].中南林业科技大学学报,2017,37(2):39-43.LI Peng,TIAN Jia,TANG Kaiwen,LUO Shuping,LI Jiang.Study on cold resistance of almond buds based on the subordinative function method[J]. Journal of Central South University of Forestry&Technology,2017,37(2):39-43.
[7] ALISOLTANI A,SHIRAN B,FALLAHI H,EBRAHIMIE E.Gene regulatory network in almond(Prunus dulcis Mill.)in response to frost stress[J]. Tree Genetics&Genomes,2015,11(5):100-103.
[8] ZHOU M Q,SHEN C,WU L H,TANG K X,LIN J. CBF-dependent signaling pathway:a key responder to low temperature stress in plants[J]. Critical Reviews in Biotechnology,2011,31(2):186-192.
[9]李科友,朱海兰.植物非生物逆境胁迫DREB/CBF转录因子的研究进展[J].林业科学,2011,47(1):124-134.LI Keyou,ZHU Hailan. Research Progress of DREB/CBF Transcription Factor in Response to Abiotic-Stresses in Plants[J]. Scientia Silvae Sinicae,2011,47(1):124-134.
[10] LACOMME C. Milestones in the development and applications of plant virus vector as gene silencing platforms[M]. Plant Viral Vectors:Springer Berlin Heidelberg,2014:89-105.
[11] BENEDICT C,SKINNER J S,MENG R,CHANG Y,BHALERAO R. The CBF1-dependent low temperature signalling pathway,regulon and increase in freeze tolerance are conserved in populus spp[J]. Plant Cell&Environment,2010,29(7):1259-1272.
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