NtIPMD gene affecting differentiation of tobacco axillary bud
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A technology of tobacco and axillary buds, applied in the field of tobacco genetic engineering
Active Publication Date: 2018-09-11
ZHENGZHOU TOBACCO RES INST OF CNTC
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In Nicotiana benthamiana, it has been reported that IPMD is involved in the acyl sugar metabolism pathway (Transcriptomic and Reverse Genetic Analyzes of Branched-Chain Fatty Acid andAcyl Sugar Production in Solanum pennellii and Nicotiana benthamiana, PlantPhysiology, 2008), but the gene in tobacco The specific function has not been reported
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Embodiment 1
[0044] This embodiment mainly affects the tobacco axillary bud differentiation NTPMD The process of gene acquisition is briefly introduced as follows.
[0045] Using the cultivar tobacco leaf as a sample, the total RNA of the tobacco leaf was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use;
[0046] Through the method of homologous comparison, refer to Arabidopsis NTPMD The sequence of the gene and the known partial gene sequence of tobacco, the designed amplification primer sequence is as follows:
[0047] F: 5'-CACCATCACGGATCCATGGCGGCTTCCTTAC-3',
[0048] R: 5'-TGGCTGCAGGTCGACTTAAACAGCAGCGGGAG-3';
[0049] Using the cDNA prepared above as a template, PCR amplification was performed using the above primers; the 50 μL amplification system was designed as follows:
[0050] Upstream primer (F primer), 1 μL;
[0051] Downstream primer (R primer), 1 μL;
[0052]cDNA template, 1 μL;
[0053] 10×buffer, 5μL;
[0054] dNTPs, 6 μL;
[00...
Embodiment 2
[0065] to be sure NTPMD Gene function in tobacco, selection NTPMD The specific nucleic acid fragment in the gene (the 68th-403rd nucleotide sequence of SEQ ID NO.1 in the sequence table) is used as a guide sequence to construct the silencer NTPMD The VIGS vector was used for the transient silencing of the gene, and the tobacco plants were further transformed to construct transgenic plants. The relevant experimental procedures are briefly introduced as follows.
[0066] (1) Construction of VIGS vector for transient silencing
[0067] First, the primer sequences for PCR amplification were designed as follows:
[0070] Perform PCR amplification (amplification length: 336bp) with the above primer sequences to obtain the guide sequence of VIGS;
[0071] Secondly, using the In-Fusion method, the above-mentioned amplified guide sequence was connected to the TRV...
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Abstract
The invention belongs to the field of tobacco gene engineering, and particularly relates to a NtIPMD gene affecting differentiation of a tobacco axillary bud. The gene comprises 1218bp basic group with the specific nucleotide sequence shown as SEQ ID NO. 1, wherein the nucleotides at the 68-403 sites are specific nucleic acid segments. Isopropyl malic dehydrogenase NtIPMD coded by the gene has thecharacteristic that isopropyl malic dehydrogenase NtIPMD comprises 405 amino acid. The primary research on the NtIPMD gene shows that the gene is related to development of the axillary bud of a plant, after the gene is silenced, development of a lateral branch can be promoted, by taking use of the characteristic, the gene can be silenced or overexpressed through gene silencing or overexpression techniques, accordingly adjustment of the plant shape can be achieved at the molecular level, meanwhile, the gene has the important effect on culturing new species of tobacco, and therefore the gene has the more significant practical value.
Description
technical field [0001] This application belongs to the field of tobacco genetic engineering, and specifically relates to a method that affects the differentiation of tobacco axillary buds. NTPMD Gene. Background technique [0002] tobacco( Nicotiana tabacum ) is an important economic crop, and tobacco leaf is its main utilization part. In the process of cultivation and growth, in order to improve production efficiency and in the later stage of tobacco leaf growth, in order to reduce the transfer of nutrients in tobacco leaves to reproductive organs, it is necessary to take "topping" operations. Although this operation destroys the apical dominance of the plant and reduces the transfer of nutrients in the tobacco leaves, it is easy to cause side branches to grow. Therefore, if the side buds are not wiped off in time, the nutrients in the leaves will also be consumed, thereby affecting the yield of tobacco leaves. Therefore, studying the mechanism of differentiation and gr...
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