Cell wall acid invertase inhibitor gene lccif and its application
An acid invertase and inhibitor technology, used in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of unclear molecular mechanism research, poor fruit setting, etc. early developmental effects
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Embodiment 1
[0022] Example 1 Cloning of litchi cell wall acid invertase inhibitor gene
[0023] 1. RNA extraction and quality detection of litchi samples
[0024] Huayueyang ultra-fast RNA extraction kit (refer to the instruction manual for the specific method) was used to analyze the leaves, flowers, pericarp, fruit pedicle, seed coat, seed kernel and other tissues of "feizixiao", "black leaf" and "nuomici" litchi The RNA was extracted, and the concentration of RNA was measured with an ultraviolet nucleic acid protein detector. Take 1 μl to measure the ratio of 260 nm to 280 nm. If it is between 1.80 and 2.00, the next experiment can be carried out. If the RNA concentration is high, it can be diluted to a certain number of times before detection.
[0025] The results of agarose gel electrophoresis figure 1 , it can be seen that RNA presents two clear bands of 28S and 18S, and the brightness of 28S is about twice that of 18S, there is no tailing phenomenon, and no DNA pollution. It sh...
Embodiment 2
[0028] Example 2 Functional verification of litchi cell wall acid invertase inhibitor gene
[0029] one, LCIF Cloning of gene fragments
[0030] Take lychees LCIF Design primers for fragments of about 500bp, and add pTRV2 before the primers Bam HI and Sma I. A 15 bp carrier sequence adapter homologous to the restriction site. The primer sequences are shown in Table 1 below. The PCR amplification conditions are 94°C, 2min; 98°C, 10s, 55°C, 30s, 72°C, 30s, 30 cycles ; 72°C, 10 min. Take 2 μl for detection by agarose gel electrophoresis, and purify and recover.
[0031]
[0032] 2. Digestion of virus silencing vector pTRV2 (pYL156)
[0033] The structure diagram of virus silencing vector pTRV1 (pYL192), pTRV2 (pYL156) is as follows image 3 , select pTRV2 (pYL156) on Bam HI and Sma Carry out double restriction endonucleases on the vector with two enzyme cutting sites, and the restriction endonuclease is selected from NEB (NewEngland Biolabs) company Bam H I HF a...
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