Rouhier, Kerry

Simmons, Hanna

English

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Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2017In Search of the t-DNA Insert: GenotypingArabidopsis Thaliana Transgenic Plant LinesHanna SimmonsKenyon CollegeKerry RouhierKenyon CollegeFollow this and additional works at: https://digital.kenyon.edu/summerscienceprogramThis Article is brought to you for free and open access by the Summer Student Research Scholarship at Digital Kenyon: Research, Scholarship, andCreative Exchange. It has been accepted for inclusion in Kenyon Summer Science Scholars Program by an authorized administrator of Digital Kenyon:Research, Scholarship, and Creative Exchange. For more information, please contact noltj@kenyon.edu.Recommended CitationSimmons, Hanna and Rouhier, Kerry, "In Search of the t-DNA Insert: Genotyping Arabidopsis Thaliana Transgenic Plant Lines"(2017). Kenyon Summer Science Scholars Program. Paper 60.https://digital.kenyon.edu/summerscienceprogram/60In	  Search	  of	  the	  t-­‐DNA	  Insert:	   Genotyping Arabidopsis	  thaliana Transgenic	  Plant	  Lines	  Background• Increasing	  global	  population,	  changing	  climate,	  and	  insufficient	  farm	  land	  means	  research	  in	  plant	  science	  is	  important• Humans	  cannot	  produce	  branched	  chain	  amino	  acids	  (BCAAs)	  like	  valine,	  leucine,	  and	  isoleucine	  though	  they	  are	  required	  for	  a	  functioning	  body,	  so	  we	  get	  them	  from	  plants• Multiple	  enzymes	  in	  the	  valine	  catabolic	  pathway	  have	  shown	  to	  be	  vital	  for	  plant	  development	  and	  growth	  (Fig.	  1)• Lack	  of	  CHY4	  is	  embryo	  lethal,	  lack	  of	  MMSD	  leads	  to	  significantly	  decreased	  germination	  rates1• HIBDH	  might	  also	  be	  significant	  in	  the	  development	  of	  plants• Goal	  was	  to	  characterize	  HIBDH	  through	  reverse	  genetics,	  taking	  away	  the	  function/production	  of	  the	  enzyme	  to	  see	  what	  effect	  that	  has	  on	  the	  plant• 5k	  bp t-­‐DNA	  sequence	  is	  inserted	  into	  the	  1.4k	  bp HIBDH	  gene	  resulting	  in	  a	  HIBDH	  transgenic	  plant	  line4• A	  complement	  plant	  line,	  one	  with	  the	  functioning	  gene	  inserted	  back	  into	  mutant	  plant	  line,	  is	  then	  developed	  to	  confirm	  phenotype	  changes	  are	  due	  to	  the	  lack	  of	  HIBDH• Isolating	  a	  homozygous	  plant	  line	  is	  the	  starting	  point	  to	  any	  further	  researchAbstractOverall	  this	  project	  set	  out	  to	  isolate	  a	  homozygous	  plant	  line	  for	  a	  mutation	  in	  the	  gene	  that	  codes	  for	  3-­‐hydroxyisobutyrate	  dehydrogenase	  (HIBDH),	  an	  enzyme	  that	  catalyzes	  one	  of	  the	  reactions	  in	  valine	  catabolism.	  Using	  a	  reverse	  genetics	  approach,	  seeds	  heterozygous	  for	  the	  mutation	  were	  planted	  and	  DNA	  was	  subsequently	  isolated	  from	  leaves.	  The	  DNA	  was	  amplified	  using	  PCR	  with	  primers	  specific	  to	  the	  gene	  of	  interest	  and	  the	  t-­‐DNA	  insert	  (the	  method	  for	  how	  the	  mutation	  was	  generated).	  Interesting	  PCR	  results	  led	  us	  to	  conclude	  that	  either	  the	  t-­‐DNA	  insert	  was	  folded	  on	  itself	  or	  inserted	  in	  duplicate.	  Either	  way	  it	  meant	  that	  we	  weren't	  going	  to	  be	  able	  to	  properly	  identify	  homozygous	  mutant	  plant	  lines.	  Concurrently,	  we	  worked	  to	  build	  a	  construct	  of	  HIBDH	  to	  create	  an	  eventual	  complement	  plant	  line.	  The	  HIBDH	  gene	  was	  cloned	  from	  A.	  thaliana and	  ligated	  into	  a	  binary	  vector	  then	  transformed	  into	  E.	  coli.	  These	  will	  then	  be	  used	  for	  another	  transformation	  into	  plants	  when	  a	  reliable	  transgenic	  plant	  line	  is	  available.	  Hannah	  Simmons	  and	  Dr.	  Kerry	  RouhierDepartment	  of	  Chemistry,	  Kenyon	  College,	  Gambier,	  OHDNA	  was	  isolated	  from	  plants	  and	  genotyped	  using	  t-­‐DNA	  and	  gene	  specific	  primers.	  Heterozygous	  plants	  give	  bands	  for	  both	  reactions,	  homozygous	  mutants	  give	  PCR	  products	  with	  only	  t-­‐DNA	  specific	  primers,	  and	  homozygous	  wild	  type	  (WT)	  give	  PCR	  products	  with	  only	  gene	  specific	  primers.	  Figure	  3.	  HIBDH	  gene	  showing	  the	  proposed	  site	  of	  the	  t-­‐DNA	  insert	  as	  well	  as	  possible	  primer	  pairings	  for	  genotyping.	  Wild	  type	  plants	  will	  show	  PCR	  products	  when	  using	  primers	  1&3	  only.	  Transgenic	  plants	  will	  produce	  PCR	  products	  when	  using	  primers	  1&2.	  Complement	  Line	  -­‐ ResultsGenotyping	  -­‐ ConclusionNone	  of	  the	  available	  primer	  pairs	  gave	  reliable	  or	  usable	  results.	  As	  in	  figure	  1,	  left	  border	  (2)	  primers	  produced	  bands	  in	  WT	  plant	  PCRs	  when	  paired	  with	  the	  expected	  gene	  specific	  primer	  (1).	  Right	  border	  (RB)	  primers	  gave	  no	  bands	  anywhere	  when	  paired	  with	  its	  associated	  primer	  (3).	  When	  the	  pairings	  were	  switched	  (i.e.	  2&3),	  the	  LB	  primers	  gave	  promising	  bands	  only	  for	  potential	  HIBDH	  knockout	  plants	  while	  RB	  continued	  to	  yield	  no	  bands.	  Products	  for	  this	  PCR	  were	  larger	  than	  expected	  so	  we	  sent	  the	  sample	  to	  be	  sequenced	  with	  poor	  results.	  To	  trouble	  shoot,	  PCR	  with	  only	  the	  LB	  primers	  was	  tried	  and	  bands	  were	  produced,	  indicating	  that	  the	  primers	  were	  binding	  in	  at	  least	  2	  places.	  This	  means	  that	  there	  are	  more	  t-­‐DNA	  inserts	  in	  the	  A.	  thaliana	  gene	  than	  originally	  thought	  or	  that	  the	  insert	  was	  folded	  in	  on	  itself,	  exponentially	  increasing	  the	  difficulty	  of	  using	  this	  plant	  strain.	  We’ve	  requested	  a	  mutant	  line	  that	  was	  recently	  characterized	  for	  further	  analysis	  in	  this	  project.Complement	  Line	  -­‐ ConclusionPCR,	  sequencing,	  and	  restriction	  digests	  suggest	  we	  have	  made	  the	  correct	  construct	  for	  a	  future	  complement	  line.	  The	  next	  step	  is	  transforming	  the	  vector	  into	  agrobacterium	  and	  then	  into	  a	  A.	  thaliana.	  The	  bacteria	  will	  insert	  their	  DNA	  into	  the	  plant	  cells	  where	  it	  will	  then	  be	  expressed	  by	  the	  plants	  and	  transferred	  to	  any	  offspring.	  Figure	  1.	  Valine	  degradation	  pathway.	  Reactions	  catalyzed	  by	  3-­‐hydroxyisobutyryl	  CoA	  hydrolase	  (CHY)	  and	  methylmalonate	  semialdehyde	  dehydrogenase	  (MMSD)	  flank	  HIBDH.	  AcknowledgementsProfessor	  K.	  Rouhier	  for	  guidance.	  This	  project	  was	  funded	  by	  the	  Kenyon	  Summer	  Science	  Scholars	  program.	  References1Gipson,	  Andrew	  B.,	  Kyla	  J.	  Morton,	  Rachel	  J.	  Rhee,	  Szabolcs Simo,	  Jack	  A.	  Clayton,	  Morgan	  E.	  Perrett,	  Christiana	  G.	  Binkley,	  Erika	  L.	  Jensen,	  Dana	  L.	  Oakes,	  Matthew	  F.	  Rouhier,	  and	  Kerry	  A.	  Rouhier.	  "Disruptions	  in	  Valine	  Degradation	  Affect	  Seed	  Development	  and	  Germination	  in	  Arabidopsis." The	  Plant	  Journal 90.6	  (2017):	  1029-­‐039.	  2Curtis,	  M.	  D.	  "A	  Gateway	  Cloning	  Vector	  Set	  for	  High-­‐Throughput	  Functional	  Analysis	  of	  Genes	  in	  Planta." Plant	  Physiology 133.2	  (2003):	  462-­‐69.	  3Hyvonen,	  Marko.	  "Guide	  to	  Expression	  Construct	  Cloning."	  (2004):	  1-­‐16.	  4Krysan,	  P.	  J.,	  Young,	  J.	  C.,	  Sussman,	  M	  R.	  "T-­‐DNA	  as	  an	  Insertional	  Mutagen	  in	  Arabidopsis." The	  Plant	  Cell	  Online 11.12	  (1999):	  2283-­‐290.5Schertl,	  Peter,	  Lennart	  Danne,	  and	  Hans-­‐Peter	  Braun.	  "3-­‐Hydroxyisobutyrate	  Dehydrogenase	  Is	  Involved	  in	  Valine	  and	  Isoleucine	  Degradation	  in	  A.	  Thaliana." Plant	  Physiology (2017):	  n.	  pag.4Figure	  5.	  PCR	  results	  with	  only	  one	  primer.	  Lanes	  1-­‐7	  correlates	  to	  different	  t-­‐DNA	  specific	  primers.	  Only	  having	  one	  primer	  should	  not	  produce	  a	  PCR	  product.1 2 3 4 5 6 7HIBDH	  mutant	  DNA1 2 3 4 5 6 7ccWT	  	  DNAGene	  specifict-­‐DNA	  specificWT heterozygousFigure	  9.	  Final	  restriction	  digest	  of	  completed	  plasmid	  in	  E.	  coli.	  1-­‐4	  are	  samples,	  5	  is	  a	  control	  of	  the	  plasmid	  without	  the	  HIBDH	  insert.	  Confirmation	  of	  step	  3	  in	  figure	  7.Figure	  2.	  Sample	  genotyping	  PCR.	  Gene	  specific	  primers	  were	  used	  in	  in	  row	  1	  and	  t-­‐DNA	  specific	  in	  row	  2.	  Bands	  only	  in	  row	  1,	  like	  in	  columns	  1-­‐5,	  indicate	  a	  homozygous	  wild	  type	  (WT)	  plant.	  Bands	  only	  in	  row	  2	  show	  a	  homozygous	  mutant	  plant	  line.	  Both	  rows	  having	  bands,	  as	  in	  columns	  6-­‐12,	  indicate	  that	  the	  DNA	  is	  heterozygous.Figure	  6.	  Arabidopsis	  thaliana	  in	  the	  growth	  room	  at	  2.5	  weeks	  old.	  Genotyping	  – Results	  Figure	  8.	  PCR	  of	  the	  restriction	  digest	  of	  both	  plasmid	  (pMDC32)	  and	  the	  insert.	  Bands	  were	  then	  purified	  and	  ligated	  together.	  Step	  one	  of	  figure	  7.Figure	  10.	  PCR	  of	  E.	  coli colonies	  that	  shows	  the	  HIBDH	  insert	  has	  been	  successfully	  transformed.	  Primers	  specific	  to	  the	  ends	  of	  the	  insert	  were	  used.	  Step	  3	  of	  figure	  7.Asc I Pac IHIBDH geneAsc I Pac IpMDC32Asc I Pac IHIBDHE. coliStep 1Step 2Step 3Figure	  4.	  PCR	  various	  primer	  parings.	  Row	  1	  is	  a	  primers	  only	  control.	  Row	  2,	  lanes	  1-­‐12	  are	  RB	  primers.	  Row	  2,	  lanes	  13-­‐16	  are	  4	  different	  templates	  with	  a	  RB	  primer	  and	  a	  new	  gene	  specific	  primer.PlasmidHIBDH	  gene	  insertFigure	  7.	  Scheme	  of	  the	  steps	  required	  for	  building	  a	  functional	  construct.	  Step	  1	  is	  isolating	  the	  AtHIBDH gene	  as	  well	  as	  choosing	  a	  vector.	  Step	  2	  is	  digesting	  then	  ligating	  the	  pieces	  together.	  Step	  3	  is	  transforming	  the	  new	  plasmid	  into	  competent	  E.	  coli	  cells5kb t-DNA1.4 kb HIBDH geneLB RB2311 & 3 => Gene specific primer pair1 & 2 => t-DNA specific primer pair 1 98765432 161514131211101 2 3 4 5 6 71 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5HIBDH	  genevectorDeveloping	  a	  complement	  plant	  line	  starts	  with	  inserting	  the	  HIBDH	  gene	  into	  a	  binary	  vector	  which	  can	  be	  transformed	  into	  E.	  coli and	  plants.	  ketoisovalerateisobutyryl-CoA3-hydroxyisobutyryl-CoA3-hydroxyisobutyratemethylmalonate semialdehydeaminotransferaseBCKDHisovaleryl-CoA	dehydrogenasemethacrylyl-CoACHYHIBDHMMSDPropionyl-CoAenoyl-CoA	HydrataseValine3 4 5 6 7HIBDH	  mutant	  DNA1 2 3 4 5 6 7WT	  	  DNA

In Search of the t-DNA Insert: Genotyping Arabidopsis Thaliana Transgenic Plant Lines

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In Search of the t-DNA Insert: Genotyping Arabidopsis Thaliana Transgenic Plant Lines

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