Capítulo IV Tendencia de la temperatura mediante un modelo armónico
4.4 Coeficiente d’ Variación lineal de la amplitud con el tiempo
The presence of the retrotransposon platforms and the i nstabi l ity of c loned fragments of these flanking regions m ade i t difficult to i solate additional sequences fl anking the cluster of ltmG, ltmM and lunK. Based on our knowledge of the genes requi red for pax i l line biosynthesi s i n P. paxilli (Young, et al . , 200 1 ; M cM i l l an , e t a l . , 2003) and aflatrem biosynthesi s in A . flavus (Zhang, et al., 2004), and the structural simil arities of these compounds to lolitrem B , orthologues of paxC, paxP and paxQ were predicted to be presen t i n N. lolii. A l i gnments of PaxC and AtmC polypeptide sequences were used to identify conserved regions to design degenerate pri mers for i solation of the N. lolii
orthologue, ltmC (Appendi x 5. 1 ). A n umber of PCR ampl ifi cation conditions w ith the fou r pri mer combinations were trialed but no evidence of an identifiable product of a
paxC orthologue was amplified from N. lolii Lp 1 9.
A t this stage of the proj ect, EST sequences from e ndophyte-infected ryegrass, which showed significant homology to paxC and paxP, were made avai lable ( by G. Spangenberg, Plant Biotechnology Centre, La Trobe U n iversity, Mel bourne) to test the hypothesi s that these EST sequences corresponded to ltm biosynthetic genes that were l in ked to the cl uster contai ning ltmG, ltmM and ltmK. A summary of these sequences w ith the m atch to their pax homologue and the region of the gene they span is shown i n Table 3.9 and Figure 3.39. Of i nterest were fragments w ith similarities to paxC and
paxP, a prenyl transferase and cytoch rome P450 monooxygenase respecti vely (Table 3 .9). The EST sequences obtai ned did not al ign to the cl uster contain i n g ltmG; ltmM
and ltmK suggesting they w ere unique . A s the qual i ty of the sequences was variable, the ESTs were a l i gned i nto conti gs usi n g the sequencher software to m i n i m i se errors for subsequent primer design. Pri mers w ere then designed to regions that w ere highly conserved to their pax homologues with a consideration on the placement of possi ble
Table 3.9 EST seq uences of potential If m genes
Gene Sequence identification ' pax gene aa rangeZ
ItmC 06nl 1 GsG 1 2 paxC 3 5- 1 74
06nl 1 FsB04
06nl 1 EsB02
06nl 1 EsG0 1
06nl1 BsC09 paxC 1 7 2-229
ItmP 06nl1 BsA06 paxP 47-304
06nl1 CsG09 RJ-J 1 5 07nl1 AsA 1 1 paxP 3 07-4 1 4 RJ-G 1 3 ItmJ 06nl 1 DsF08 paxpS 4 1 4-49 5 RJ-N 1 7
E value3 % identit
i
Primers42e-2 6 60% 101 1 89/ 1 90
1 e-0 7 49% 101 1 90/1 9 3
1 e-40 3 6% 101 1 9 1 / 1 9 2
4e- 1 9 44% 101 1 9 2 / 1 9 5
7e-0 5 3 8% 10120 5/206
' The EST identification number of the sequences provided by G. Spangenberg ( Plant Biotechnology Centre, La Trobe University, Melbourne) and R. Johnson (AgResearch, Palmerston North).
2The amino acid range of the match of the EST sequences to the pax homologue.
3The E-value of the BLASTX match or the % identity to the pax homologue
4Primers used to amplify a genomic Lp 1 9 fragment
sThe best match was to a P450 m onooxygenase from Asperillus nidulans (AN 1 598), at 3e-09
PCR size 3 60 644 3 74 603 242
1 101 1 89
50
1 00
1 50
200
250
300
EsB02 BsC09EsG01
FsB04
GsG1 2
92 1011 98 1011 91 1011 95 101205 101 1 94 1 2 3 5B
06P a x P
50
1 00
1 50
200
250
300
350
400
450
500
RJ-J 1 5
RJ-G 1 3
DsF08BsA06
AsA 1 1
RJ-N1 7CsG09
ItmJ ItmPFigure 3.39 Schematic d iagram of PaxC and Pax P showing placement of the EST seq uences
Schematic diagram of (A) PaxC and (8) PaxP, showing the relative placement of the EST sequences. The polypeptide sequence is represented as green blocks with the size indicated in amino acid residues underneath. The intron placements are numbered above the polypeptide. The primers used for PCR amplification are positioned above the region used for primer design. The EST sequences that are part of the ItmP or the ItmJ gene are shown as lines below the EST positions. The EST identification numbers (Table 3.9) have been reduced to the last five numbers.
conserved i ntrons between the ltm and pax genes. Additional EST sequences were made available (by R. Johnson, AgResearch, Pal merston North) from a suppressi on subtraction hybridisation technique, w here three transcri pts, RJ-J 1 5 , RJ-G 1 3 and RJ
N I 7, were identified as genes up-regul ated in N. lolii Lp 1 9-i nfected perennial ryegrass
when compared to uni nfected perennial ryegrass. These three EST sequences were a l so incl uded i n the sequence analysis data (Table 3.9; Fig. 3. 39).
The contig of EST fragments, 06n l l GsG 1 2, 06n l l FsB04, 06nl l EsB02 and 06n l l EsG0 1 , was desi gnated ltmC based on their significant match to PaxC using the BLASTX algorithm (Table 3.9). A fragment of 360 bp, the same size as that of the c DNA, was ampl ifi ed from Lp 1 9 genomic DNA using pri mers 101 1 89 and 101 1 90. This confi rmed that the EST fragments were of fu ngal ori gin. PCR amplification w as used to join the ltmC conti g to 06nl l BsC09 a potential ltmC fragment (Table 3.9; Fig. 3. 39). Ampl ification from N. lolii Lp 1 9 genomic DNA w ith pri mers 101 1 90 and 101 1 93 gave a 644-bp product that was 77-bp l arger than the pred icted transcri pt size, indicating the presence of an intron in thi s gene ( Fig. 3. 39). The PCR product generated from Lp 1 9 genomic DNA with primers 101 1 90 and 101 1 93 was sequenced and compared to the EST seq uences for confirmation of the i ntron.
The contig of EST fragments, 06n l l BsA06 and 06n l l CsG09 (Fig. 3. 39), had a sign ifi cant match to paxP, span ning amino acid resid ues 84 through 304 (Table 3 .9). Pri mers, 1 01 1 9 1 and 101 1 92, designed to this sequence ampl ified a 374-bp fragment from
N. lolii Lp 1 9 genomic DNA. This confi rmed these EST sequences were of fun gal origin. The size of the genomic fragment was 60 bp larger than the predicted cbNA product i ndicati ng that this gene contains at least one i ntron.
EST seq uences with the BLASTX matches to paxP aligned i nto three i ndependent contigs (Table 3.9; Fi g. 3. 39). Conti g 1 contai ned EST sequences 06n l l BsA06, 06n l l CsG09 and subsequently RJ-J l 5 , contig 2 contai ned EST sequences 07nl l AsA I 1 and RJ-G 1 3 , and conti g 3 contai ned EST seq uences 06nl l DsF08 and RJ- N 1 7. PCR was pelformed to test w hether these three contigs were part of a si ngle cytochrome P450 monooxygenase gene or were i n fact multiple genes. Ampl ification of N. lolii
Lp 1 9 genomic DNA w ith pri mers 101 1 92 and 101 1 95 l i nked contigs 1 and 2 and therefore these two contigs are a part of the same fun gal cytochrome P450 monooxygenase gene subsequently n amed ltmP. The PCR fragment generated from N.
loW Lp l 9 genomic DNA w ith pri mers 101 1 92 and 1 01 1 95 was sequenced and compared to the EST data for confi rmation of the i ntron. Contig 3 contai ned the primer-bi nding site for pri mer 101 1 94 and this primer would not ampl ify a PCR product from Lp 1 9 genomic DNA when pai red with pri mer 101 1 92. This contig was therefore considered an i ndependent cytochrome P450 monooxygenase fragment and was subsequently
named ltmJ. Pri mers, 101205 and 101206, were designed to the conti g seq uence of ltml.
These pri mers ampl ified a 242-bp fragment from Lp l 9 genomic DNA and confirmed
that ltmi was of fungal ori gin.
Hybridi sation of an ltmC fragment to genomic di gests of the endophyte strai ns Lp 1 9, Fi t , and E8 show s that this fragment is only pre sent i n the lol itrem-prod ucing strains, Lp 1 9 and Fl l ( Fi g. 3.40A ). The [tmC hybridisation pattern of each di gest w as identi cal between the Lp l 9 and Fl l strains, but the hy bridi sation signal i n Lp 1 9 was twice that of Fl l despite identical DNA loadi ngs.
Two ltmP fragments were used as hybridi sati on probes to di gested genomic DNA of strai ns N. loW Lp 1 9, E. Jestucae FI I and E. typhina E8. Each fragment was only detected in the Lp 1 9 and Fl l strains ( Fig. 3.40). The ltmP fragment ampl ified wi th pri mers 101 1 9 1 and 101 1 92 gave different Lp 1 9 and FI ! banding patterns with each restriction enzyme ( Fig. 3.40C). The !tmP fragment ampl ifi ed with pri mers 1 01 1 96 and 1 01 1 98 spans both a HindI I I and Sst! restriction enzyme site ( Fig. 3.4 1 ). Hy bridi sation w ith thi s fragment resulted in two Hind l l l and SstI hybridising bands. T he 0.5-kb
Hind I I I and 9 kb SstI fragments in each di gest were the same size in the Lp 1 9 and Fl I di gested DNA ( Fig. 3 .408). The 9-kb SstI fragment hybridised to both the ltmP
fragment, ampl ified w ith pri mers 101 1 96 and 101 1 98 , and to the ltmC fragment. These data show that the ltmC and limP genes are l i n ked.
The ltmi fragment hybridised to the lol itrem-producing strai ns N. loW Lp 1 9 and E.
festucae FI I ( Fi g. 3.40). This fragment hy bridised to a � 1 8 kb Lp 1 9 SstI fragment, a
band of the same size as seen with the ltmP probes suggesti ng l i n kage of ltmJ to ltmP.
The presence of the three EST fragments, ltmC, ltmP and ltmi, correlated with strains known to produce i ndole-diterpenes. None of the fragments hybridi sed to genomic di gests of E8, a l ol itrem non-producing strain. This pattern of hybridisation w as used to identify the previous ltm cluster contai n i ng ltmC, ltmM and ltmK, and therefore, complete seq uence surround i ng the genes lunC, ltmP and ltmJ was obtai ned.