Composite
Part:BBa_K4887022
Designed by: Duoqing LIN Group: iGEM23_Shanghai-BioX (2023-09-19)
Expression vector of IbGBSSI knockout system
This part is the expression vector used for knockout gene IbGASSI (BBa_K4887001) in sweet potato (Ipomoea batatas).
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 2106
Illegal PstI site found at 3528
Illegal PstI site found at 3732
Illegal PstI site found at 3762
Illegal PstI site found at 4974 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 2106
Illegal PstI site found at 3528
Illegal PstI site found at 3732
Illegal PstI site found at 3762
Illegal PstI site found at 4974 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1567
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2106
Illegal PstI site found at 3528
Illegal PstI site found at 3732
Illegal PstI site found at 3762
Illegal PstI site found at 4974 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2106
Illegal PstI site found at 3528
Illegal PstI site found at 3732
Illegal PstI site found at 3762
Illegal PstI site found at 4974
Illegal NgoMIV site found at 1157
Illegal NgoMIV site found at 1176
Illegal NgoMIV site found at 2394
Illegal NgoMIV site found at 3498
Illegal NgoMIV site found at 3571
Illegal NgoMIV site found at 4056
Illegal NgoMIV site found at 4965
Illegal AgeI site found at 6940 - 1000COMPATIBLE WITH RFC[1000]
13.5 Results:
(1) Construction of the expression vector of sgRNA (IbGBSSI)
The validated backbone vector of sgRNA (IbGBSSI) was digested by EcoR I & Hind III and inserted into the corresponding sites of the binary vector pCAMBIA1301s, harbouring the Hygromycin B resistance gene HygR and the reporting gene GUS, and obtained the expression vector of sgRNA (IbGBSSI): psgR-Cas9-sgRNA(IbGBSSI)-p1301s.(2) Agrobacterium tumefaciens transformation
The expression vector of sgRNA (IbGBSSI) was then transferred into Agrobacterium tumefaciens LB4404 by reeze-thaw method. The positive transformants containing were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin (Fig. 1). The obtained clones were validated by performing PCR detection for the sgRNA sequence with primers M13F/oligo2 (The sequence of M11F was: 5’-TGTAAAACGA CGGCCAGT-3’). The gel electrophoresis results (Fig. 2) showed that the gene band were approximately 100bp, as expected, indicating that expression vector of sgRNA (IbGBSSI) was been constructed and transferred into Agrobacterium tumefaciens successfully.
Fig. 1 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s
Fig. 2 PCR result of sgRNA (IbGBSSI) in A. tumefaciens transformed with expression vector of sgRNA (IbGBSSI)
(3) Genetic transformation of sweet potato
Embryogenic calli of sweet potato ware infected with the A. tumefaciens transformants containing expression vector of sgRNA (IbGBSSI) by co-culture on the MSD media containing hygromycin B and cefalexin (Fig. 3-A). After selection with hygromycin B, positive transformed calluses were obtained (Fig. 3-B). These calli were further cultured to obtain transgenic sweet potato seedlings (Fig. 4).
Fig. 3 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of postively transformed callus with hygromycin (B)
Fig. 4 Seedlings of IbGBSSI-knockout lines after preliminary selections
(4) Verification of transgenic sweet potato plants
1) GUS detection
The GUS gene, is a commonly used reporter gene harboured in pCAMBIA1301s. Its expression product β-glucuronidase is a hydrolase that can catalyze the hydrolysis of many β-glucoside esters. It can decompose X-Gluc into blue substances, to observe the expression of foreign genes in transgenic plants and identify transgenic plants.After the regenerated seedlings grew leaves, GUS staining was performed on these transgenic plants. Two successfully transformed sweet potato lines were preliminarily screened and designated as 23216004 and 23216005, whose leaves turned green (Fig. 5).
Fig. 5 Results of GUS staining
2) PCR detection
Genome DNA of these two transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using two pairs of primers which were designed based on the Cas9 protein gene and the hygromycin resistance gene (HygR), respectively. As the result, the transgenic lines 23216004 and 23216005 were further validated (Fig. 6).The sequences of the two pairs of primers were as bellow:
- CAS9-F: 5’-atggactataaggaccacgacgg-3’; CAS9-R: 5’-ttgtcgcctcccagctgagacag-3’
- HygR-F: 5’-Atgaaaaagcctgaactcac-3’; HygR-R: 5’-ctatttctttgccctcggac-3’
Subsequently, these two transgenic lines and the wild-type B23 were planted with the method of cuttage in an experimental greenhouse to harvest starch-rich tubers.
Fig. 6 Detection results of PCRs for gene HygR and gene Cas9.
3) Determination of the expression level of IbGBSSI in root tubers
Two months after transplantation, the tubers of the transgenic lines were harvested. It showed that the number and size of the root tubers were largely consistent with the wild type (Fig. 7).
Fig. 7 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse
Fig. 8 Q-PCR result of the relative expression level of IbGBSSI in root tubers
(5) Starch analysis of transgenic sweet potato root tubers
Freshly harvested sweet potato tubers were cleaned, peeled, and sliced into small pieces. Starch was extracted from these pieces for qualitative and quantitative detection afterwards.
1) Qualitative detection of the starch components
When exposed to iodine, Amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type (Fig. 9). It indicated that the total starch of the transgenic lines was composed mainly of amylopectin.
Fig. 9 Component detection of total starch by iodine staining
1) Qualitative detection of the starch components
When exposed to iodine, Amylose appears blue, while amylopectin appears reddish brown or purple red. Therefore, the component qualitative detection of the total starch from the transgenic lines was performed. As the result, the total starch of the transgenic lines appears reddish brown while that appears blue of the wild type (Fig. 9). It indicated that the total starch of the transgenic lines was composed mainly of amylopectin.
Fig. 9 Component detection of total starch by iodine staining
Fig. 9 Component detection of total starch by iodine staining
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