Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean

Prepared By:- Bishnu Adhikari Plant Resources Development Lab

Student Id No: 2016226596

25th Oct. 2016

Outlines:

• Introduction• Objectives • Materials and Methods• Results and Discussions• Conclusion

Saponin: Plant constituent which bring about frothing in an aqueous solution. Saponins = Glycosides, hydrolyzed by acids to give an aglycone and different sugars related to uronic acids. • Aglycone called sapogenin (insoluble in water).Structure of the

sapogenin determines the type of saponin (steroidal or tetracyclicsteroidal or tetracyclic triterpenoid & pentacyclic types).

• Both types of glycosides have a glycosidal linkage at C- 3& common biogenic origin (mevalonic acid & isoprenoid units).

Introduction:

Fig. Structure of Saponin

Gene Silencing • Turning off a gene and happens naturally• Occurs at a transcriptional or a post-transcriptional level• An important laboratory technique • Epigenetic regulation of gene expression• Differs with gene knock out

RNA interference(RNAi) Post transcriptional gene silencing Entry of double-stranded RNA (dsRNA) Small double-stranded fragments by Dicer enzyme RNAi induced silencing complex (RISC); Argonaute proteins Cleavage or translational repression of the mRNA molecules makes the

genes inactive.

Fig. RNA interference(RNAi)

Objectives:

• Manipulation of saponin content & composition for the improvement of seed quality.

• Functional analysis of saponin in soybean.• Use of RNAi in transgenic soybean plants.• Obtaining transgenic seeds in which biosynthesis of saponin

will be suppressed.

Materials and Methods:Reverse transcription & Polymerase Chain Reaction analysis

Soybean plant cultivar “Jack” RNeasy Plant Mini Kit QuantiTect Rev. Transcription Kit

Transformation vectors: pUHR:11S-IR RNAi vector Amplifying promoter subunit gene

Soybean transformation: Somatic embryos were induced from immature cotyledons of cv. Jack cultured on MSD40 medium & maintained in FNL medium.

Fig. Soybean Cv. Jack

Fig: Schematic representations of two β-amyrin synthase genes of soybean

Fig. RT-PCR analysis of β-amyrin synthase gene expression in soybean tissues

Southern blot analysis:Investigate the presence & integration pattern of transgenes in T1 plantsHybridization probe, a 586-bp fragment of linker sequenceInverted sequences of GmBAS1 was amplified by PCR with the primers 50.

LC–MS/MS analysis of saponins: Supernatants of the crude extracts analyzed by tandem mass spectrometry with an HCT Ultra ion-trap mass equipped with an electrospray ionization source & coupled to a liquid chromatography system.

Fig. Liquid Chromatography Mass Spectrometry (LC/MS)

.

Fig. Phylogenic relations among GmBAS2 & GmBAS1 as well as other β -amyrin synthases.

Transgenic T3 plants harboring either pUHR:BAS-RNAi-a or pUHR:BAS-RNAi-b & the control transgenic line were grown under the green house condition. Abundance of β -amyrin synthase (GmBAS1 and GmBAS2) mRNA was determined with MGB probe.

Fig. Structures of the RNAi vectors pUHR:BAS-RNAi-a and pUHR:BAS-RNAi-b.

Fig. Alignment of the cDNA sequences inserted into the RNAi vectors.

Results and Discussions:

• β -Amyrin synthase gene profiling in soybean GmBAS1 was highly expressed in tissues , whereas the expression of GmBAS2 was more restricted and marked lower level. GmBAS1 is the predominant β -amyrin synthase in soybean plants.

• Generation of transgenic soybean with RNAi vectors targeting β -amyrin synthase

Four ( a4-2, a5-2, a6-1, and a6-2) and two ( b2-5 and b2- 7) transgenic lines were selected

T1 plants obtained for the 6 transgenic lines exhibited distinct banding patterns in Southern blot analysis with a probe targeted to the RNAi cassette

Fig. Detection of transgenes in BAS-RNAi transgenic soybean plants by Southern blot analysis.

Fig. Separation and detection of saponin components in transgenic seed cotyledon.

Fig: Separation and detection of saponin components in transgenic seed hypocotyls.

• Depletion of saponins of β -amyrin synthase expression in transgenic soybean seeds

Fig. Impaired accumulation of saponins in transgenic T1 seeds.

Fig. Impaired accumulation of saponins in transgenic T3 seeds.

Depletion of saponins………….

Fig. Suppression of β -amyrin synthase

• Suppression of β -amyrin synthase expression in transgenic soybean seeds

The reduction in saponin content in seeds of the RNAi soybean lines was due to suppression of β -amyrin synthase gene expression.

• Antioxidant activity of transgenic soybean seeds

Table. Effect of saponin deficiency on the antioxidant activity of transgenic soybean seeds

Oxygen Radical Absorption Capacity values didn’t differ significantly with compare to control transgenic seeds. Saponins don’t contribute substantially to the antioxidant activity of soybean seeds.

Conclusion:

• GmBAS1 was highly expressed in various soybean tissues, whereas no or only a low level of GmBAS2 expression was detected under the normal growth condition.

• RNAi vectors for GmBAS1 gave the higher level of β -amyrin synthase gene expression including the developing seeds & it shares high level of homology with GmBAS2.

• Two different RNAi levels resulted in sufficient suppression of the mRNA levels to enable a marked reduction in the saponin content of soybean seeds.

• Seed saponins aren’t required for normal growth and development in soybean.

THANK YOU FOR KIND ATTENTION!