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

1. 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

2. Outlines:
• Introduction
• Objectives
• Materials and Methods
• Results and Discussions
• Conclusion

3. 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

4. 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.

5. Fig. RNA interference(RNAi)

6. 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.

7. 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

8. Fig: Schematic representations of two β-amyrin synthase genes of soybean
Fig. RT-PCR analysis of β-amyrin synthase gene expression in soybean tissues

9. Southern blot analysis:
Investigate the presence & integration pattern of transgenes in
T1 plants
Hybridization probe, a 586-bp fragment of linker sequence
Inverted 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)

10. .
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.

11. 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.

12. 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

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

14. 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

15. Fig. Impaired accumulation of
saponins in transgenic T1 seeds.
Fig. Impaired accumulation of
saponins in transgenic T3 seeds.
Depletion of saponins………….

16. 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.

17. • 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.

18. 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.

19. THANK YOU FOR KIND
ATTENTION!