Pear hexose transporter gene pbht1 and its application
A transporter and gene technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of no research reports, unclear effect on the function of pear monosaccharide transporter, etc., and achieve the effect of increasing the hexose content
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Embodiment 1
[0033] Example 1 Cloning of PbHT1 Gene of Pear
[0034] Using the pulp of'Ya Pear' 40-100 days after blooming as the test material, total RNA was extracted and reverse transcribed, and the obtained first strand cDNA was used to amplify the PbHT1 gene. Use CTAB method (CTAB extraction buffer: 2% CTAB, 2% PVP K-30, 0.05% spermidine, 10mM Tris·HCl (pH=8.0), 25mM EDTA, 2M NaCl) to extract total RNA, take 1μg RNA sample After incubating at 37°C for 30min with 1U DNaseI (purchased from TaKaRa), adding 1μL EDTA (25mM) and incubating at 65°C for 10min. The TOYOBO reverse transcription kit (purchased from TakaRa Company, according to the kit instructions for the synthesis of the first strand cDNA. The amplification primers are: forward primer PbHT1-F1: 5'-TCCCAAGAACCCTAACAATGCC-3' (SEQ ID NO.3 ); Reverse primer PbHT1-R1: 5'-TGGGAACAATAATATTCAGTGCTACC-3' (SEQ ID NO.4). 25μL PCR reaction system includes: 1×PCR buffer (purchased from TakaRa), 2.5mM MgCl 2 (Purchased from TakaRa company), 0....
Embodiment 2
[0037] Example 2 Changes in PbHT1 gene expression and sugar content during pear fruit development
[0038] 1. qRT-PCR analysis of PbHT1 gene in pear fruit development
[0039] The methods of extracting total RNA from pear pulp and cDNA synthesis are the same as in Example 1. Using pear β-tubulin (AB239681) as an internal control, the nucleotide sequence of the primer is as follows: forward primer TUB-F: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO. 5), reverse primer TUB-R: 5' -CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 6). Using Primer Premier5.0 to design gene-specific qRT-PCR primer pairs in the open reading frame of the PbHT1 gene, the nucleotide sequence of the primers is as follows: forward primer PbHT1-F2: 5'-CCTCTGCGTGGCAATCGTCAT-3' (SEQ ID NO. 7), the reverse primer PbHT1-R2: 5'-TTCTCCAGGGTTCCCATCCAC-3' (SEQ ID NO. 8).
[0040] qRT-PCR uses SYBR Green kit (purchased from TaKaRa company, operated according to kit instructions). The 20μL qRT-PCR reaction system includes: 10μL 2×SYBR Premi...
Embodiment 3
[0044] Example 3 Subcellular localization of PbHT1 gene
[0045] In this example, the onion epidermis was used to study the subcellular location of the PbHT1 gene. The expression vector used was pCAMBIA1302, and the expression vector had the GFP gene ( Figure 4 ). Use RT-PCR to amplify the entire ORF of PbHT1 gene, design amplification primers that include the ORF sequence and remove the stop codon, and add BglII and SpeI to the 5'ends of the positive and negative primers. , The amplification primer with restriction site is obtained: the nucleotide sequence of the forward primer PbHT1-F3 is: 5'-GA AGATC TATGCCTGCTGTTGGTAT-3' (SEQ ID NO.9), the nucleotide sequence of the reverse primer PbHT1-R3 is: 5'-GG ACTAGT TGGGAACAATAATATTCAGTGCTACC-3' (SEQ ID NO. 10). The underline is the restriction site, AGATCT Is the BglⅡ restriction site, ACTAGT SpeI restriction site. First install the amplified product on the pMD19-T vector to obtain the recombinant vector PMD19-T B / S -PbHT1. Si...
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