Cloning And Expression Analysis Of The Seed Germination Related Mannanase Gene From Solanum Rostratum

Buffalobur(Solanum rostratum Dunal) is a noxious and invasive weed species native to North America. Due to its wide adaptability and strong competitiveness, this species has spread into many regions in China and caused a serious threat to local biodiversity. Seeds are responsible for the occurrence, dispersal and infestation of weeds. Buffalobur seeds exhibit deep dormancy and it is difficult for one time control and thorough eradication of established populations. To explore the molecular mechanisms in regulating seed dormancy and germination in buffalobur, two genes encoding endo-beta-mannanase, the key enzyme in degradation of endosperm cell wall during seed germination, were cloned from buffalobur. Relative gene expressions during seed imbibition and germination process under application of exogenous hormones were then analyzed. Furthermore, the prokaryotic gene expression vectors were constructed and the function of fusion proteins was ascertained by gel diffusion method. The results were as follows.1. Two mannanase related genes were cloned from buffalobur. The full-length gene sequence of Sr MAN1 was 1921 bp, while Sr MAN2 was 3075 bp. The full-length coding sequence of Sr MAN1 was 1143 bp, which encoded a protein of 380 amino acids. The predicted molecular weight and isoelectric point of Sr MAN1 were 42.9 k D and 5.4, respectively. The full-length coding sequence of Sr MAN2 was 1242 bp, which encoded a protein of 413 amino acids. The predicted molecular weight and isoelectric point were 46.6 k D and 5.2, respectively.2. q RT-PCR results showed that the expression of Sr MAN1 and Sr MAN2 reached its peak level when the length of radicle was about 10 mm and the length of hypocotyl was close to 2 mm under GA treatment. The relative expression of Sr MAN1 and Sr MAN2 could hardly be detected during seed imbibition under ABA treatment, while both genes experienced increase at first and then decrease under GA and water treatment, and the peak value of expression appeared earlier under GA treatment. The peak expression of Sr MAN1 under GA was higher than that of under water, while the peak expression of Sr MAN2 under GA was lower than that of in water. It might not be significant tissue and time specificity in the expression profile of Sr MAN1 and Sr MAN2. GA could influence the expression level of Sr MAN1 and Sr MAN2, but the effect on Sr MAN2 was smaller than that on Sr MAN1.3. Gene expression vectors p ET-32a-Sr MAN1 and p ET-32a-Sr MAN2 were successfully constructed and transformed into E. coli BL21(DE3) to induce expression of Sr MAN1 and Sr MAN2. The SDS-PAGE analysis showed that tag fusion protein of Sr MAN1 and Sr MAN2 were close to the predicted molecular weight, 62.78 k D and 66.37 k D, respectively. The gel diffusion test showed that when incubation solution of positive strains was added to the mannans-containing gel, significantly enlarged hydrolytic circles could be seen after a period of incubation. This confirmed the activity of mannanase in the fusion proteins.