Yang Jinyu, Wang Runying, Ren Na, Zhao De-Gang, Huang Xiaozhen, Zhao Yichen
College of Life Sciences, College of Tea Sciences, The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025, China.
Plant Conservation and Breeding Technology Center, Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Science, Guiyang, 550006, China.
Sci Rep. 2024 Dec 5;14(1):30398. doi: 10.1038/s41598-024-81695-2.
Eucommia ulmoides (E. ulmoides) is a valuable gum-producing plant and traditional Chinese medicine. The utilization value of E. ulmoides varies according to the sex of the plant, and due to its perennial characteristics, the identification of male and female is challenging. To meet production demands, gender selection through grafting has been employed. The fusion of rootstock and scion cells in grafted plants can be enhanced by β-1, 4-glucanase, thus improving the grafting survival rate. However, extracting β-1, 4-glucanase in vivo poses difficulties. In this study, the β-1, 4-glucanase gene of E. ulmoides was cloned, and the total length of the gene was 1917 bp, encoding 638 amino acids. Pichia pastoris engineering bacteria were used to express β-1, 4-glucanase. The optimal fermentation conditions included a pH of 6, a temperature of 28 ℃, a methanol content of 1.5%, and a fermentation period of 96 hours. After purification, the enzyme activity of the target protein was measured to be 286.35 U/mL. Protein concentrations of 5 mg/mL, 10 mg/mL, and 15 mg/mL were tested for E. ulmoides grafting. The results showed that the protein could promote wound healing and improve the survival rate of E. ulmoides grafting. In conclusion, this study successfully developed an enzyme that improves the survival rate of E. ulmoides grafting, and provides valuable insights for the breeding of male and female E. ulmoides grafts.
杜仲是一种珍贵的产胶植物和传统中药。杜仲的利用价值因植株性别而异，且由于其多年生的特性，鉴别雌雄株具有挑战性。为满足生产需求，已采用通过嫁接进行性别选择的方法。β - 1,4 - 葡聚糖酶可增强嫁接植株中砧木和接穗细胞的融合，从而提高嫁接成活率。然而，在体内提取β - 1,4 - 葡聚糖酶存在困难。在本研究中，克隆了杜仲的β - 1,4 - 葡聚糖酶基因，该基因全长1917 bp，编码638个氨基酸。利用毕赤酵母工程菌表达β - 1,4 - 葡聚糖酶。最佳发酵条件为pH值6、温度28℃、甲醇含量1.5%、发酵周期96小时。纯化后，测得目标蛋白的酶活性为286.35 U/mL。对杜仲嫁接试验了5 mg/mL、10 mg/mL和15 mg/mL的蛋白浓度。结果表明，该蛋白可促进伤口愈合，提高杜仲嫁接成活率。综上所述，本研究成功开发出一种提高杜仲嫁接成活率的酶，并为杜仲雌雄嫁接育种提供了有价值的见解。
