Populus deltoides*Populus nigraPdHARDY gene and application thereof
A gene, European and American technology, applied in the field of PdHARDY gene, to achieve the effect of improving expression
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Embodiment 1
[0020] The extraction of embodiment 1 total RNA
[0021] 1. The European and American Populus 107 material used in the present invention is taken from Baoding, Hebei. It is an annual cutting cuttage cultivated in the nursery of Beijing Forestry University. After the cuttage, it is watered once every three days in order to keep the soil moist. After new leaves grow, the growth similar (Growing for 3 months) European and American poplar seedlings were sprayed with 250mM NaCl, and the top leaves were removed at 0, 2, 4, 6, and 12 hours respectively, placed in liquid nitrogen, and then stored in a -80°C ultra-low temperature refrigerator for later use. For total RNA extraction and cDNA synthesis.
[0022] 2. The process of extracting total RNA from the leaves of Populus americana under 250mM NaCl stress by CTAB method:
[0023] (1) Before extracting RNA, add DTT at a final concentration of 0.1 mM to 2×CTAB buffer, and preheat at 65°C.
[0024] (2) Put 0.5 g of liquid nitrogen gr...
Embodiment 2
[0030] Example 2 Using fluorescent quantitative PCR method to detect the expression of PdHARDY gene of Populus americana in adversity
[0031] According to the conserved sequence of PdHARDY gene of Populus americana, the upstream primer of specific primers: PdHARDY1 (SEQ ID NO.1) and downstream primer: PdHARDY2 (SEQ ID NO.2) were designed.
[0032] Primer name
sequence name
Base sequence (5'--3')
PdHARDY1
SEQ ID NO.1
ATGCAAGATT ATCAACAAAG CAGAATC
PdHARDY2
SEQ ID NO.2
TCAAGGGAATTTCCAAAGGTTCTGGTC
[0033]According to the method of Example 1, the cDNA samples prepared by extracting the apical leaves at 0, 2, 4, and 6 hours were used as templates, and PdHARDY1 (SEQ ID NO.1) and PdHARDY2 (SEQ ID NO.2) were used as templates. Primers were used for fluorescent quantitative PCR analysis.
[0034] The fluorescent quantitative PCR reaction system (20 μL):
[0035]
[0036] The fluorescent quantitative PCR reaction is divided int...
Embodiment 3
[0038] Example 3 Clone PdHARDY gene of Populus americana by PCR method
[0039] According to the conserved sequence of PdHARDY gene of Populus americana, the upstream primer of specific primers: PdHARDY1 (SEQ ID NO.1) and downstream primer: PdHARDY2 (SEQ ID NO.2) were designed.
[0040] Primer name
sequence name
Base sequence (5'--3')
PdHARDY1
SEQ ID NO.1
ATGCAAGATT ATCAACAAAG CAGAATC
PdHARDY2
SEQ ID NO.2
TCAAGGGAATTTCCAAAGGTTCTGGTC
[0041] The synthetic cDNA prepared by the method described in Example 1 was used as a template, and PdHARDY1 (SEQ ID NO.1) and PdHARDY2 (SEQ ID NO.2) were used as primers for PCR reaction;
[0042] The PCR reaction system is (25 μl):
[0043]
[0044]
[0045] The PCR reaction program is as follows: 35 cycles of pre-denaturation at 95°C for 5 min, each cycle of denaturation at 95°C for 50 s, annealing at 63°C for 90 s, extension at 72°C for 2 min, and finally, the sample was extended at...
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