Transferred The Seed Storage Protein Genes Of Psathyrostachys Huashanica Into Triticum Aestivum And Studied On Its Quality Effects And Its Molecular Cloning

Psathyrostachys huashanica Keng. (2n = 2x = 14, NsNs) is an endemic species in China that has been listed as an endangered and imperatively protected wild species. It is characterized by early maturity and resistance to drought, salinity, wheat take-all fungus and stripe rust. In this paper, a wheat-P. huashanica 1Ns disomic addition line"H9021-28-5"was developed and its chromosomal configuration and behaviors were analyzed, and the high molecular weight glutenin subunit (HMW-GS), low molecular weight glutenin subunit (LMW-GS) and gliadin genes of P. huashanica were identified. The agronomic traits, the quality effect, and the mineral element content of this alien addition line were surveyed. The HMW-GS, LMW-GS and gliadin genes of P. huashanica were molecular cloned and the sequence were analyzed. Main results are as follows:1) The Cytogenetics investigations revealed that the chromosome number and configuration of"H9021-28-5"were 2n = 44 = 22 II. The mitotic and meiotic GISH analysis indicated that"H9021-28-5"contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The SDS-PAGE and A-PAGE analysis showed that"H9021-28-5"carried the HMW-GS, LMW-GS and gliadin genes of P. huashanica. The results suggest that these storage protein genes of P. huashanica had been transferred into common wheat, and"H9021-28-5"is a wheat-P. huashanica 1Ns disomic addition line.2) The agronomic traits survey on H9021-28-5 indicated that P. huashanica 1Ns chromosome may be have a reduced effect on the wheat plant height, length of spike, No. of spikelets, and grains per spike, and may be have a stimulating effect on grain weight, length, diameter, and hardness. The processing quality analysis indicated that H9021-28-5 have been significantly improved in the sedimentation value, protein content, dough development time, stability time, extention resistance, and dough extensibility. Determination of mineral elements shows that many kinds of element content in P. huashanica is higher than wheat, and 11 kinds of element content in alien addition line H9021-28-5 have been markedly enhanced, compared with wheat cv. 7182.. The SSR and EST-SSR markers analysis showed that, there are four pairs of SSR primers and six pairs of EST-SSR primers can be preliminary used as P. huashanica 1Ns chromosome-specific molecular markers. 3) The HMW-GS promoter of P. huashanica were cloned by PCR method, and its sequences were analyzed. Analysis on this promoter gene HGp-Ns-1 (GQ139524) showed that the sequence contain an E-box, N-box, G-box, HMW-endosperm-specific 38bp enhancer and TATA-box. Phylogenetic analysis indicated that HGp-Ns-1 has a relatively closer relationhip with the y-type HMW-GS promoter gene of Ps. spicata.4) The four LMW-GS gene of P. huashanica were isolated, and sequences analysis showed that these four sequences all included signal peptide, N-terminal region, the repetitive domain, and C-terminal region, successively. There are eight cysteine residues in coding region. Starting as METSRIPSL- or METSRIPGL- in N-terminal domain indicated that these 4 sequences are the m-type LMW-GS gene. FJ600493 (LG-Ns-1), FJ600494 (LG-Ns-2) and GQ223386 (LG-Ns-3) may be pseudogene because of two premature stop codons appeared in coding sequences. Structural analysis on GQ139528 (LG-Ns-3) revealed that it contained a part of LMW-GS promoter gene and coding region of LMW-GS. Promoter sequence analysis showed that there have the conservative endosperm box, E-box, F-box, CAAT box, and TATA box. Phylogenetic analysis suggested that these four P. huashanica LMW-GS genes have a relatively closer relationship with the E. ciliaris LMW-GS genes.5) The fourα-type and aω-type gliadin gene sequences were isolated from P. huashanica genomes. Sequences structural analysis on fourα-gliadins revealed that these sequences contain signal peptide, N-terminal repetitive domain, polyglutamine domain I, unique domain I, polyglutamine domain II, and unique II, successively. Sequence Gli-Ns-2 (FJ713595) may be a pseudogene because of two premature stop codons appeared in unique domain I. There 8 or 9 cysteine residues in the four sequences. Sequence structure analysis on aω-gliadin gene Gli-Ns-1 (FJ600500) showed that it have signal peptide, N-terminal region and part of repetitive domain of typical structure ofω-gliadin, and it may be a pseudogene because of a premature stop codons appeared in repetitive domain. Phylogenetic analysis suggested that FJ713595 have closer kinship withα-gliadin genes of leymus mollis and E. ciliaris. Sequence Gli-Ns-4 (GQ139525), Gli-Ns-4 (GQ139526), Gli-Ns-5 (GQ139527), and FJ600500 may be a new type of prolamin gene family.These reseach are of important significance for enriching and improving wheat resources of storage proteins genes, and for continuing to exploit the advantageous genes of the endangered species P. huashanica.