Part:BBa_K4887001

IbGBSSI

The encoding gene of granule-bound starch synthase I (GBSSI) in sweet potato (Ipomoea batatas). It is one of the starch biosynthetic pathway genes on the starch biosynthetic pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1459
  Illegal BsaI.rc site found at 123
  Illegal SapI site found at 1156
  Illegal SapI.rc site found at 1225
  

Results

(1) GBSSI-sgRNA design

The selected sgRNA is located 70 bp downstream of the start codon, with a total sequence length of 27 bp (Fig. 1). sgRNA oligos were obtained by annealing with the designed primers following: Oligo 1 (GBSSI-sgRNA1): 5’-GTGGGGTTGGGTCAATTAGCCCTGAGGAGC-3' Oligo 2 (GBSSI-sgRNA2): 5’-AACTGGTGGACTTGGAGATGTTCTTGGAGG-3’

Fig. 1 The target region of gene IbGBSSI and the location of the gRNA. Exons are shown as square frames and surrounding introns appear as lines. sgRNA and PAM are highlighted in yellow and green, respectively.

(2) The expression level of IbGBSSI in storage roots

The relative expression level of the gene IbGBSSI was determined with the method of Quantitative Real-time PCR with the root-tuber cDNA as templates. The result showed that the relative expression level of IbGBSSI in root tubers of the transgenic lines (0.1063 and 0.2407) was much lower than that of the wild type (1.0000) (Fig. 2). It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.

Fig. 2 Q-PCR result of the relative expression level of IbGBSSI in root tubers