GbAnn9 gene for improving quality of cotton fiber and application
A cotton fiber and protein technology, applied in the field of peptides, can solve the problem of no data on transgenic cotton
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Embodiment 1
[0075] Cloning of cDNA fragment of embodiment 1 Gbann9 gene
[0076] (1) Synthesis of cDNA from sea-island cotton germ epidermis
[0077] The construction method of the library was carried out by using the cDNA library construction kit of Stratagene Company (Stratagene, CA, USA), and the specific steps of the method were as follows.
[0078] The total RNA of cotton tissues was extracted from immature embryos of sea-island cotton variety Pima-90 3 days after flowering. Weigh 0.5g of sea-island cotton immature embryos, grind them into fine powder with liquid nitrogen, and divide them into two 1.5ml eppendorf tubes. Add 1ml to each tube (Invitrogen, CA, USA) Shake vigorously to make the mixture uniform, and place at room temperature for 5 min. Then centrifuge at 12,000g for 10min at 4°C, and pipette the supernatant into a clean 1.5ml eppendorf tube. Add 0.2ml chloroform to each tube, vibrate vigorously for 15s, and place at room temperature for 2-3min. Then centrifuge at 12...
Embodiment 2
[0088] The artificial synthesis of embodiment 2 GbAnn9 gene
[0089] According to the completed nucleotide sequence containing the 951bp coding region, firstly divide it into 4 segments and synthesize single-stranded oligonucleotide fragments with a length of about 150-200bp and sticky ends according to the sequences of the positive strand and the secondary strand respectively. Anneal the 6 complementary single-stranded oligonucleotide fragments with one-to-one correspondence between the positive strand and the auxiliary strand respectively to form 6 double-stranded oligonucleotide fragments with sticky ends. Mixed double-stranded oligonucleotide fragments, T 4 DNA ligase catalyzes the assembly into a complete GbAnn9 gene. The synthetic DNA fragment contains the 46-987 nucleotide sequence in SEQ ID NO: 1, and the two ends of the synthetic gene contain XbaI and SacI sites.
[0090] The above-mentioned artificially synthesized 5' and 3' end restriction sites are XbaI and SacI ...
Embodiment 3
[0091] Example 3 Construction of GbAnn9 Gene Plant Expression Vector
[0092] The specific method of GbAnn9 gene plant expression vector construction is as follows:
[0093] A. pBI121 and pCAMBIA201 were digested with HindIII and EcoRI, and the pBI121 fragment with p35S-GUS-Nos-ter was ligated into pCAMBIA2301 to form the intermediate vector p35S-2301-GUS;
[0094] B. Use XbaI and SacI to cut p35S-2301-GUS and the above-mentioned artificially synthesized GbAnn9 gene, replace the GUS at the corresponding restriction site of p35S-2301-GUS with GbAnn9, thereby obtaining the GbAnn9 gene plant expression vector ( Such as figure 1 shown). Then it was transformed into Agrobacterium strain LBA4404 for transforming cotton.
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