Isolation And Analysis Of The CDNA Sequences For Blue Endosperm In Wheat

Two kinds of cDNA libraries were constructed using the blue-grained wheat monosomic addition line: one was a conventional cDNA library, while the other was a suppression subtractive hybridization (SSH) library. Some blue endosperm related cDNA sequences were isolated. The purpose of this study was to understand the genetic basis of blue endosperm development in wheat and further explore the related genes.The results can be summarized as follow:1. The conventional cDNA library from blue wheat seeds were constructed following the method described by SMART cDNA library construction manual. The obtained unamplified library contained about 1.2×10~6 clones. The titer of the partial amplified library was 2×10~9 pfu/ml.2. The suppression subtractive hybridization library for wheat blue endosperm was constructed. The SSH library contained about 10,000 individual recombinant clones with the cDNA inserts ranging from 100 to 500 base pairs (bp) and the average size was around 300bp.3. A total of 16 positive clones were obtained by screening the SSH library. Sequence homologous querying indicated that:(1) Nine cDNA sequences had homologues in Genbank. Among them, three showed high homologous with wheatα-gliadin genes and one with wheat Glu-B3 gene. Because of their higher expression levels, these genes might be artifactually enriched during SSH library construction. The rest five cDNA sequences had high homology with wheat mRNA for serpin, Zea mays P450 monooxygenase, puroindoline genes for wheat grain hardness, and two unknown sequences from wheat and rice, respectively. These five cDNA sequences might be differentially expressed genes related to blue endosperm.(2) No homologues were found for the rest of seven differentially expressed cDNA sequences in Genbank. These cDNA sequences may be related to the development of blue endosperm and probably the novel genes. 4. Northern blot analysis indicated that four cDNA sequences showed blue-endosperm specific expression manner, for example, the cDNA sequence 3 that had high homologous with Zea mays P450 monooxygenase, and the cDNA sequence 15 with unknown function, showed higher expression level in blue seeds compared with in white one, whereas the cDNA sequence 14 and 16 only expressed in blue wheat seeds. The latter two cDNA sequences may be highly correlated to the development of blue endosperm.