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利用RACE-PCR技术首次从药用真菌猪苓中分离得到猪苓菌核的4个小GTPase基因。这4个基因分别命名为Pu Rho A1、Pu Rho A2、Puypt1及Pu Ras,Pu Rho A1全长698 bp,编码区共585 bp,编码194个氨基酸,推测分子量为21.75 k D,理论等电点为6.44;Pu Rho A2全长837 bp,其中编码区长585 bp,可编码194个氨基酸,计算其分子量为21.75 k D,理论等电点为6.33;Puypt1为一条896 bp的c DNA,编码区为615 bp,可编码204个氨基酸,经计算分子量为22.556 k D,理论等电点为5.75。Pu Ras的c DNA全长为803 bp,其中编码区长639 bp,编码212个氨基酸,推测分子量为23.821 k D,理论等电点为5.2。Pu Rho A1具有法尼酰转移酶催化位点(FT-Caa X)和香叶烯基转移酶催化位点(GGT1-Caa X),而Pu Rho A2仅具有香叶烯基转移酶催化位点(GGT1-Caa X);Puypt1具有小GTPase家族中Rab1-Ypt1的完整保守结构域;Pu Ras具有法尼酰转移酶催化位点(FT-Caa X)。系统进化树结果显示猪苓的4个小GTPase都隶属于担子菌类群。实时荧光定量PCR分析结果表明在菌核形成初期Puypt1、Pu Ras、Pu Rho A1在菌核中表达量显著高于菌丝,而Pu Rho A2在菌核中的表达量显著低于菌丝,说明这4个小GTPase基因可能参与了猪苓菌核的形态发生。
For the first time, four small GTPase genes were isolated from Polyporus umbellatus with RACE-PCR. These four genes were named Pu Rho A1, Pu Rho A2, Puypt1 and Pu Ras, respectively. Pu Rho A1 was 698 bp in length and encoded a total of 585 bp encoding a polypeptide of 194 amino acids with a predicted molecular weight of 21.75 kD. The theoretical isoelectric point Was 6.44. Pu Rho A2 was 837 bp in length, encoding a polypeptide of 194 amino acids with a length of 585 bp. The calculated molecular weight was 21.75 kD and the theoretical isoelectric point was 6.33. Puypt1 was an 896 bp cDNA. The coding region Is 615 bp, encoding 204 amino acids. The calculated molecular weight is 22.556 kD, and the theoretical isoelectric point is 5.75. Pu Ras c DNA was 803 bp in length, encoding a 639 bp region encoding 212 amino acids, suggesting a molecular weight of 23.821 kD and a theoretical isoelectric point of 5.2. Pu Rho A1 has the farnesyltransferase catalytic site (FT-Caa X) and the sesquiterpene transferase catalytic site (GGT1-Caa X), while Pu Rho A2 has only the site of the hemeenylase transferase enzyme ( GGT1-Caa X); Puypt1 has a complete conserved domain of Rab1-Ypt1 in the small GTPase family; and Pu Ras has a farnesyl transferase catalytic site (FT-Caa X). Phylogenetic tree analysis showed that all four small GTPases of Polyporus umbellatus belong to basidiomycetes. The results of real-time PCR showed that the expression of Puypt1, Pu Ras and Pu Rho A1 was significantly higher in the sclerotia than that in the mycelia at the initial stage of sclerotia, while the expression level of Pu Rho A2 in sclerotia was significantly lower than that of the mycelium These four small GTPase genes may be involved in the morphogenesis of Polyporus umbellatus.