Application of AaBAM3 gene in improving cold resistance of kiwi fruits
A technology for kiwifruit and cold resistance, applied in the field of genetic engineering, can solve problems such as unknown effects, and achieve the effect of improving cold resistance
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Embodiment 1
[0031] Embodiment 1 AaBAM3 gene cloning
[0032] RNA was extracted from the branches of Actinidia jujuba 'Kuilu', and the single-stranded cDNA was obtained as a template by a reverse transcription kit (Toyobo Co., Ltd.), and the full-length sequence of AaBAM3 gene was obtained by PCR amplification. PCR amplification results see figure 1 , a band with a size of 1643bp was obtained.
[0033] The primer sequences used are as follows:
[0034] AaBAM3-F: 5'-ATGGCTTTGGCGCTACATTC-3'; SEQ ID NO.1;
[0035] AaBAM3-R: 5'-CAGTAGAGCAGCCTCCTTCG-3'; SEQ ID NO.2;
[0036] The PCR reaction system is 25 μl: ddH 2 O 18.25 μl, 10×PCR buffer 2.5 μl, dNTP 1.0 μl, AaBAM3-F 1 μl, AaBAM3-R 1 μl, cDNA 1.0 μl, ExTaq DNA Polymerase (5 U / μl) 0.25 μl. The PCR reaction program was 94°C: 5min, 95°C: 30s, 60°C: 30s, 72°C: 90s, 35 cycles, 72°C: 10min, 4°C: hold.
[0037] The target fragment obtained by PCR amplification was cloned through the pMD-18T vector, and then ligated to the Pbi121-GFP vector (pu...
Embodiment 2
[0042] AaBAM3 gene expression analysis in 'Kuilu' kiwifruit, 'Kuilu' kiwifruit seedlings were treated at a low temperature of 4 ℃ for 0h, 6h, 1d, 3d, 5d, and the expression of AaBAM3 gene was detected by qRT-PCR. Select Actin as an internal reference gene, use 2 -△△Ct The formula calculates the relative expression of genes, and the results are shown in figure 2 .
[0043] The primer sequences used for qRT-PCR detection are as follows:
[0044] AaBAM3-Q-F: 5'-ACCTACCAATAGCGCGGATG-3'; SEQ ID NO.5;
[0045] AaBAM3-Q-R: 5'-AACTAACCCTTCTGGCGAGC-3'; SEQ ID NO.6;
[0046] Actin-F: TGCATGAGCGATCAAGTTTCAAG; SEQ ID NO.7;
[0047] Actin-R: TGTCCCATGTCTGGTTGATGACT; SEQ ID NO.8.
[0048] It was found that the expression of AaBAM3 gene increased gradually with the increase of the low temperature treatment time at 4°C.
Embodiment 3
[0049] Embodiment 3 AaBAM3 gene functional verification
[0050] In order to study whether AaBAM3 gene plays a role in the process of resisting low temperature stress in kiwifruit, its function was identified through the analysis of transgenic kiwifruit.
[0051] Actinidia sinensis 'Hongyang' was used for the functional verification of AaBAM3 gene, and the transformation of kiwifruit was carried out according to the method provided by Wang et al. (2008). The 30-day-old 'Hongyang' tissue culture seedlings were used as the material, and the medium was MS solid medium with pH 5.8. The culture environment is 25°C, 14h light, 10h dark.
[0052] (1) Screening of transgenic kiwifruit plants: The sequenced Pbi121-AaBAM3 vector plasmid was transformed into Agrobacterium EHA105 by a liquid nitrogen freeze-thaw method. Adjust the concentration of Agrobacterium bacterium OD=0.6-1.0, infect and transform 'Hongyang' by the leaf disk method, and see for the regenerated plants image 3 . ...
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