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:

10
INCOMPATIBLE 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
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INCOMPATIBLE 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
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INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1567
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INCOMPATIBLE 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
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INCOMPATIBLE 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
1000
COMPATIBLE 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

RNA was extracted from the fresh root tubers and cDNA was obtained through reverse transcription later. Then, 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. 8). It revealed that the knock-out of IbGBSSI in the pathway of starch synthesis was successful.

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

2) Quantitative detection of the starch composition

Firstly, standard starch solutions with different gradients of amylose content were prepared (Table 1) and their absorbance values at a wavelength of 620nm, OD620, were obtained. Subsequently, using the absorbance values as the vertical axis and the amylose content as the horizontal axis, the standard curve representing the linear relationship was plotted between the content of branch starch and OD620 and the following calculation formula was fitted (where y represents the content of amylose in total starch (%), and x represents OD620) (Fig. 10):

Fig. 10 Standard curve for amylose content (%) of total starch

Later, starch solutions were prepared respectively from total starch extracted earlier from root tubers of the two genotypes and the wild type and their OD620 values were determined, with three replicates for each line. After plugging the obtained OD620 values into formula (4), the amylose content was quantified in each sample.
Compared to the amylose content of the wild type (23.3306%), the amylose content in the total starch of the two transgenic lines was only 0.5657% and 0.7613%, respectively (Table 2). According to the study by Wang (2019), knockout of IbGBSSI had no impact on the total starch content in sweet potato tubers. It indicated that sweet potato lines of high-content amylopectin synthesis have been developed with the knock-out of the gene IbGBSSI by using a highly efficient CRISPR/Cas9 system.

Fig. 20 Amylose content (%) of total starch of root tubers

Part:BBa_K4887022