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| Extraction of Total RNA and Cloning and Expression Analysis of UrSTR Gene from Uncaria rhynchophylla |
| MU De-Tian1, WAN Ling-Yun2, WEI Shu-Gen2, WU Chang-Qiao1, PAN Li-Mei2, LIU Yi-Song3, TIAN Yi1, FU Jin-E2,*, TANG Qi1,* |
1 College of Horticulture, Hunan Agricultural University, Changsha 410128, China;
2 Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China;
3 College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China |
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Abstract Uncaria rhynchophylla is one of commonly used staple Chinese medicinal materials in China, mainly used for headache, Alzheimer's disease and hypertension. Its active ingredients are mainly terpenoid indole alkaloids (TIAs). Strictosidine synthase (STR) is a key enzyme in the biosynthesis pathway of terpenoid indole alkaloids. So far, it has not been reported in U. rhynchophylla. In order to search for STR genes involved in the TIAs biosynthesis of U. Rhynchophylla. LC-MS method was used to measure the content of rhyncholphylline and isorhyncophylline in root, leaf, stem hook and capsule of U. rhynchophylla. Total RNA from different parts of U. rhynchophylla were compared by 4 extraction methods. UrSTR gene, which may be involved in the biosynthesis of rhyncholphylline and isorhyncophylline, was cloned and bioinformatics analysis was carried out. The expression patterns of UrSTR in different parts of U. rhynchophylla were detected by qPCR. The results were as follows: (1) The contents of rhyncholphylline and isorhyncophylline were the highest in capsules and the lowest in roots through LC-MS method. (2) The improved CTAB method was the best extraction method for different parts of U. rhynchophylla. (3) The total length of UrSTR was 1 122 bp, encoding a peptide chain of 374 amino acids with a transmembrane helical structure. The theoretical molecular weight of UrSTR was 41.446 kD, and the theoretical isoelectric point was 7.549. The evolution of UrSTR gene was clustered with Coffea arabica and C. canephora, showing a close genetic relationship. (4) The results of qPCR showed that UrSTR gene was mainly expressed in capsule and the lowest expression was found in root. The analysis showed that the expression of UrSTR gene was consistent with the trend of the content of rhyncholphylline/isorhyncophylline, indicating that UrSTR gene may be one of the candidate genes for U. rhynchophylla involved in the biosynthesis of TIAs. This study provides reference for the functional verification of UrSTR gene and the analysis of U.rhynchophylla alkaloid synthesis pathway.
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Received: 24 September 2021
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Corresponding Authors:
*tangqi@huanu.edu.cn;duanwei3014@163.com
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