Vector construction and functional analysis of AcCBF1 using VIGS method in almond flower organs

Abstract: 【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.