Studies On The Function Of Heterotrimeric G Protein In Brassica Napus

Heterotrimeric G protein signaling is the most significant and conservative signal transduction pathway in eukaryotes. Major components of heterotrimeric G protein known so far are Ga, Gβ, Gy subunits and the regulator of G protein signaling protein (RGS). Ga separates from βγ-dimer after G protein is activated and reunites with βγ-dimer with the effect of RGS protein. Gβ and Gy subunits work in the form of βγ-dimer. RGS protein accelerates the rate of GTP hydrolysis of Ga subunit and terminates the G-protein signaling.In the present study, we used Yangyou6 and transgenic lines to investigate the subcellular localization, interaction between Ga, Gβ, Gy subunits and RGS protein, roles in ABA response and quality formation and possible mechanism of G proteins in Brassica napus. The main results were as follows:(1) We identified and characterized BnGA1,BnGB1, BnGG2 and BnRGSl genes. The full length of BnGAl contained an open reading frame of 1152bp encoding a putative 20 kDa polypeptide with a predicted theoretical isoelectric point of 5.83. It doesn’t cross membrane and has a Ga domain. The full length of BnGB1 contained an open reading frame of 1137 bp encoding a putative 57 kDa polypeptide with a predicted theoretical isoelectric point of 6.66. It doesn’t cross membrane and has seven classic Gp WD40 domains. The full length of BnGG2 contained an open reading frame of 303 bp encoding a putative 17 kDa polypeptide with a predicted theoretical isoelectric point of 4.69. It doesn’t cross membrane and has a Gγ domain. The full length of BnGAl contained an open reading frame of 1380 bp encoding a putative 52.6 kDa polypeptide with a predicted theoretical isoelectric point of 8.43. It has seven putative transmembrane domains in the N-terminal and RGS box in the C-terminal.(2) We analyzed subcellular localization of the components of G protein using transient expression of onion epidermal cells and tobacco leaf. The results showed that BnGA1 was located on the membrane; BnGB1 was located on the membrane in the shape of dots; BnGG2 was located on the membrane and in the nucleus; BnRGS1 was located on the membrane.(3) We investigated interactions between the components of G protein using the mating-based split-ubiquitin system. The results showed that there were interactions between BnGB1 and BnGA1, BnGG2 and BnGA1, BnGA1 and BnRGS1, BnGG2 and BnGB1, BnRGS1 and BnGB1. There was no interaction between BnGG2 and BnRGS1.(4) We optimized the protocols of protoplast extraction and plasmid transformation in Brassica napus. The results showed that the components of G protein may be involved in Brassica napus response to plant hormone signaling.(5) Compared with wild type Yangyou6, the essential fatty acid and protein contents were increased while glucosinolate content was decreased, and the expression of the key enzyme genes in essential fatty acid synthesis fad2, fad3, fad6, fad7, fad8 were increased. The results indicated that Gα subunit and RGS protein played a negative role in the enzyme system of fatty acid synthesis in order to affect the systhsis process of fatty acid.