Populus deltoids*populus nigra basic-domain leucine-zipper (PdbZIP) gene and application thereof
A gene, European and American technology, applied to the PdbZIP gene and its application field, to achieve the effect of improving expression
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Embodiment 1
[0020] Example 1 Extraction of 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, and 6 hours respectively and 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 ground mat...
Embodiment 2
[0030] Example 2 Using fluorescent quantitative PCR method to detect the expression of PdbZIP gene of Populus americana in adversity
[0031] According to the conserved sequence of PdbZIP gene of Populus americana, specific primers were designed: upstream primer: PdbZIP1 (SEQ ID NO.1) and downstream primer: PdbZIP2 (SEQ ID NO.2).
[0032] Primer name
sequence name
Base sequence (5'--3')
PdbZIP1
SEQ ID NO.1
ATGAGCCGCA TCTTCACAAC TCCTGAA
PdbZIP2
SEQ ID NO.2
TTAGCCTAGC AAATCTGAAG AACTTGTAAT
[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 PdbZIP1 (SEQ ID NO.1) and PdbZIP2 (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 into three s...
Embodiment 3
[0038] Example 3 Clone PdbZIP gene of Populus americana by PCR method
[0039] According to the conserved sequence of PdbZIP gene of Populus americana, specific primers were designed: upstream primer: PdbZIP1 (SEQ ID NO.1) and downstream primer: PdbZIP2 (SEQ ID NO.2).
[0040] Primer name
sequence name
Base sequence (5'--3')
PdbZIP1
SEQ ID NO.1
ATGAGCCGCA TCTTCACAAC TCCTGAA
PdbZIP2
SEQ ID NO.2
TTAGCCTAGC AAATCTGAAG AACTTGTAAT
[0041] The synthetic cDNA prepared by the method described in Example 1 was used as a template, and PdbZIP1 (SEQ ID NO.1) and PdbZIP2 (SEQ ID NO.2) were used as primers for PCR reaction;
[0042] The PCR reaction system is (25 μl):
[0043]
[0044] 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 72°C for 10 min. Th...
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