Gene for regulating and controlling length of cotton fiber and application thereof
A cotton fiber and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0027] Example 1: Construction of recombinant vectors and genetic transformation mediated by Agrobacterium
[0028] 1. Design and synthesize tRNA and sgRNA at the editing site of the GhHD7 gene (its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2);
[0029] The sequence of tRNA (SEQ ID NO.3) is: 5'-AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA-3';
[0030] The sequence of sgRNA (SEQ ID NO.4) is: 5'-GATAATGCGTGAGAGTTTGG-3'.
[0031] 2. Connect the sgRNA sequence in series with the tRNA to the pRGEB32-Cas9 gene editing vector. The constructed recombinant vector is shown in the figure figure 1 shown.
[0032] 3. After the recombinant vector was constructed and transferred into Escherichia coli, primers pG32-U6-F and pG32-Ubi2-R were used for PCR verification and sequencing ( figure 2 As shown, the gray part is the GhHD7 targeting site), after successful sequencing, the plas...
Embodiment 2
[0035] Example 2: Detection of GhHD7 knockout cotton T2 generation Cas9 fragment and detection of editing site
[0036] 1. Get the seeds harvested from the T0 generation positive plants in Example 1, plant them, and obtain the T1 generation plants, then in the T1 generation strains, harvest the seeds and plant them, and obtain the GhHD7 gene knockout cotton T2 generation plants (hereinafter referred to as the T2 generation) .
[0037] The T2 generation and WT (R15) were sampled. The sampling site was young leaves. There were 3 lines in total, 285 individual plants. DNA was extracted and amplified with Cas9 fragment-specific primers (shown in Table 1). The amplification system As shown in Table 2, the amplification program is: 94°C, 5min; 94°C, 30s, 55°C, 30s, 72°C, 1min, 35cycle; 72°C, 10min; 4°C, ∞. Then electrophoresis detection, the result is as follows image 3 As shown, it can be seen from the test results that the T2 generation was separated, some plants contained the ...
Embodiment 3
[0048] Embodiment 3: the comparison of T2 generation and wild plant ovule
[0049] 1. Take the ovules on the day of flowering (0DPA) of the WT and T2 generations and fix and dehydrate them, dry and spray gold, and then take pictures with a scanning electron microscope ( Figure 7 ), the field of view is 200 times, and the scale is 200um. Preliminary observation showed that the ovule density of WT was higher than that of T2 generation.
[0050]2. Further compare statistical data of 3 repetitions of 3 strains of WT and T2 generations. Take 281, 282, and 284 T2 gene knockout lines of 3 replicates and take pictures. Each scanning electron microscope photo randomly selects 3 parts, the area is 100um×100um, and the statistical results are T-tested. The results are as follows: Figure 8 As shown, it can be seen that the number of ovules per unit area of the three lines of the T2 generation was significantly lower than that of the WT.
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