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Title: Functional analyses of cis-acting elements from SPORAMIN
promoter and investigation the SNPs between SPORAMIN genes
MS student: Hsiao-Chi Chang
Advisor: Dr. Kai-Wun Yeh
Abstract：
SPORAMIN is a storage protein of sweet potato tuber, and the expression of
sporamin gene is wound-inducible in leaves. In order to define the
wound-response cis-acting elements of sporamin promoter, the genomic
walking method was used to clone upstream promoter regions. Three
segments of promoter region of this gene family were obtained. Although the
sequences of these promoters are various, they all contain almost the same
cis-acting elements, such as W-box and GCC-box. Therefore, the expression
pattern of each gene member may be quite similar. Our previous data
demonstrated that a sporamin promoter : SP1 (PROSPOA) is wound-induced
in the transgenic tobacco by promoter-GUS fusion assay. The GUS staining
results indicate that the sequences contain the NOS-like element confer the
most wound-induced activity, the SP8a and G-box also may contribute to
enhance the effect of NOS-like element. So we use the synthetic promoter
method to identify each cis-acting element. Constructions with one, two and
four copies of each element were tested its wound-induced activity by
promoter-GUS fusion assay. We also tested the constructs with mixing motifs
in transgenic Arabidopsis. The GUS staining and GUS quantitative results
showed that NOS-like element and SP8a motif both contribute to the GUS
expression under mechanical wounding in transgenic Arabidopsis. The
NOS-like element and SP8a motif indeed confer wound-induced activity when
working alone, but the expression level of reporter gene is higher according
with the number of motif increased. Although the induced level of GUS proteins
were different between these constructs, the expression patterns were quite
similar. The induced GUS proteins were expressed in wounded leaf, root, stem,
and unwounded leaves. So the wounding response might be a systemic effect.
We also constructed cDNA library of sweet potato tuberous root to clone more
sporamin genes, because we intend to investigate the SNPs (single nucleotide
polymorphisms) between these genes. 20 sporamin genes were cloned, 19 of
these genes belongs to sporamin A and only one gene belongs to sporamin B.
The identity between these two groups is 80%, and the identity within each
group is about 90%. We can also divide sporamin A into two sub-family, and
the identity within each sub-family is 95%. The sequence polymorphisms are
taking place almost in the 5’, and 3’-UTR, these findings are resemble with the
theory proposed by Hattori in 1989. We found that the active domain within
amino acid sequences of these sporamin genes were identical, so the activity
of these proteins might be the same. The evolution prediction of this gene
family tends to purifying
evolution pathway.