Evidencias de ciudad educadora en los tres cruces.

The sequences of the three TR-ACS genes; TR-ACS1, TR-ACS2, and TR-ACS3; isolated from the Tienshan ecotype (designated TR-ACS-T), as reported in this thesis, and those previously isolated from genotype 10-F (designated TR-ACS-10F) were compared for identity at the nucleotide and amino acid level. The TR-ACS4-T

sequence was not compared as it is the novel TR-ACS gene and its sequence has not yet been reported previously from other white clover varieties.

The alignment comparisons between nucleotide and amino acid sequences of TR- ACS1-T and TR-ACS1-10F and TR-ACS2-T and TR-ACS2-10F are shown in Appendices XII and XIII. The partial sequences of the two TR-ACS1 genesconsist of 629 bp or 210 amino acids in the reading frame, and they share 96% identity at nucleotide level and 93% amino acid identity. The two TR-ACS1 genes share high identity in the conserved regions and both have the conserved amino acid residues (eight in TR-ACS1-T and nine in TR-ACS1-10F) required for the binding of the substrate to PLP. For the nucleotide and deduced amino acid sequences of the TR- ACS2 genes, the partial sequence comparison shows that TR-ACS2-T and TR-ACS2- 10F consist of a 642 and 645bp coding region, respectively, and they share 98% homology at the nucleotide level and 92% amino acid identity (Appendices XIV and XV).

Results 2

137 In common with the TR-ACS1 and TR-ACS2 genes, both TR-ACS3 sequences comprise the partial coding frame region within box I to VI. The coding frame sequence of TR-ACS3-T comprises 622 bp nucleotides which is approximately the same length as TR-ACS1-T, TR-ACS2-T and TR-ACS4-T. However the TR-ACS3- 10F sequence only comprises 562 bp which is 60 bp smaller and translates into 20 missing amino acids (Figures 4.10 and 4.11). The aligned nucleotide and amino acid sequence of these two TR-ACS3 show that the TR-ACS3-10F sequence is missing 20 of the amino acids that occur in conserved domain six (the reactive site of the enzyme). This reactive site is present in TR-ACS3-T and in the three other TR-ACS- T proteins (see Section 4.1.2.). Deletion of these 20 aa from TR-ACS3-10F means that only 6 out of 11 (3 of these, including the catalytic lysine residue, are in domain VI) of the conserved amino acid residues that are required for the binding of pyridoxal 5’phosphate are present (Metha et. al., 1989). With the exception of the deleted 20 aa, identity values of the two TR-ACS3 genes are very high (more than 90%). Therefore, TR-ACS3-T and TR-ACS3-10F represent the same isoform, but only TR-ACS3-T occurs as an active ACC synthase.

Results 2 138 TR-ACS3-T ATTGCCAGAATTCAGAAATGCTGTGGCAAATTTCATGTCAAAAGTGAGAGGTGGTAGGGT 60 TR-ACS-10F ATTGCCAGAATTCAGAAATGCTGTGGCAAATTTCATGTCAAAAGTGAGAGGTGGTAGGGT 60 ************************************************************ TR-ACS3-T AAGATTTGATCCTGACCGTATATTGATGAGTGGTGGAGCAACAGGGGCAAATGAATTAAT 120 TR-ACS-10F AAGATTTGATCCTGACCGTATATTGATGAGTGGTGGAGCAACAGGGGCAAATGAATTAAT 120 ************************************************************ TR-ACS3-T CATGTTCTGTTTGGCTGGTCCTGGTGATGCCTTTTTGGTTCCTAGCCCTTATTATCCAGC 180 TR-ACS-10F CATGTTCTGTTTGGCTGATCCTGGTGATGCCTTTTTGGTTCCTAGCCCTTATTATCCAGC 180 ***************** ****************************************** TR-ACS3-T ATTTGTTCGTGATTTGTGTTGGARAACCGGTGTGCAACTAATTCCTGTCCAATGTCATAG 240 TR-ACS-10F ATTTGTTCGTGATTTGTGTTGGAGAACCGGTGTGCAACTAATTCCTGTCCAATGTCATAG 240 *********************** ************************************ TR-ACS3-T CTCAAACAATTTCAAGATAACAAGAGAAGCACTTGAAGAAGYTTATWTGAAAGCACAAGA 300 TR-ACS-10F CTCAAACAATTTCAAGATAACAAGAGAAGCACTTGAAGAAGCTTATATGAAAGCACAAGA 300 ***************************************** **** ************* TR-ACS3-T AAGAAACATCAATGTGAAAGGGTTAATCATAACAAATCCATCAAACCCTCTAGGAACAAC 360 TR-ACS-10F AAGAAACATCAATGTGAAAGGGTTAATCATAACAAATCCATCAAACCCTCTAGGAACAAC 360 ************************************************************ TR-ACS3-T AATAGAAAAAGAAACACTAAAGAGCATAGTTAGTTTCATAAATGAAAACAACATTCATTT 420 TR-ACS-10F AATAGAAAAAGAAACACTAAAGAGCATAGATAGTTTCATAAATGAAAACAACATTCATTT 420 ***************************** ****************************** TR-ACS3-T AGTGTGTGACGAAATCTATTCCGGCACAGTTTTCAACACTCCGAAATACGTAAGTGTCGC 480 TR-ACS-10F AGTGTGTGATGAAATCTATTCCGGCACAGTTTTCGACACTCCGAAATACGTAAGTGTCGC 480 ********* ************************ ************************* TR-ACS3-T CGAAGTTATACAAGAAATGGAAGAATGCAAAAAAGACCTCATTCATATCATATATAGTTT 540 TR-ACS-10F CGAAGTTATACAAGAAATGGAAGAATGCAAAAAAGACCTCATTCATAT--- 528 ************************************************ TR-ACS3-T ATCAAAAGACATGGGACTTCCGGGTTTCAGAGTCGGTTTAGTTTATTCGTACAATGATGA 600 TR-ACS-10F ---GTACAATGATGA 540 ************ TR-ACS3-T AGTTGTGAATTGCGGTCGAAAA 622 TR-ACS-10F AGTTGTGAATTGCGGTCGAAAA 562 **********************

Figure 4.10 Alignment of nucleotide sequences of the TR-ACS3 genes isolated

from the Tienshan ecotype (TR-ACS3-T) (this thesis) and that previously

isolated from genotype 10-F of the cultivar Grasslands Challenge (TR-ACS3- 10F, as reported by Murray and McManus, 2005). (*) represents identical

Results 2

139

2 3

TR-ACS3-T LPEFRNAVANFMSKVRGGRVRFDPDRILMSGGATGANELIMFCLAGPGDAFLVPSPYYPA 60 TRACS310F LPEFRNAVANFMSKVRGGRVRFDPDRILMSGGATGANELIMFCLADPGDAFLVPSPYYPA 60 *********************************************.**************

4 TRACS3T FVRDLCWITGVQLIPVQCHSSNNFKITREALEEAYMKAQERNINVKGLIITNPSNPLGTT 120

TRACS310F FVRDLCWRTGVQLIPVQCHSSNNFKITREALEEAYMKAQERNINVKGLIITNPSNPLGTT 120 ******* ****************************************************

5

TRACS3T IEKETLKSIVSFINENNIHLVCDEIYSGTVFNTPKYVSVAEVIQEMEECKKDLIHIIYSL 180 TRACS310F IEKETLKSIDSFINENNIHLVCDEIYSGTVFDTPKYVSVAEVIQEMEECKKDLIHM---- 176 ********* *********************:***********************: 6

TRACS3T SKDMGLPGFRVGLVYSYNDEVVNCGRK 207 TRACS310F ---YNDEVVNCGRK 187

***********

Figure 4.11 Alignment of deduced amino acid sequences of the TR-ACS3 gene isolated from the Tienshan ecotype (TR-ACS3-T) and the TR-ACS3 sequence previously identified from genotype 10-F of the cultivar Grasslands Challenge (TR-ACS3-10F, as reported by Murray and McManus, 2005).

(*) represents identical residue, (.) represents semi-conserved substitution sequence; (:) represents conserved substitution sequence (-) represents no sequence; and () represents conserved residues found in aminotransferase and other ACC synthases while boxed-italics represent the catalytic residue of the enzyme. Shaded, numbered sequences represent the conserved regions of ACC synthase and shaded underlined sequences represent the active site of the enzyme.

The high identity at the nucleotide and amino acid levels confirmed that the RT-PCR based gene cloning successfully generated ACC synthase genes from the Tienshan ecotype. The identity values of these six TR-ACS genes are summarized in Table 4.7.

Results 2

140 Table 4.7 Nucleotide homology and amino acid identity values of six ACC synthase genes identified in the Tienshan ecotype and genotype 10F of the cultivar Grasslands Challenge

Nucleotide level TR-ACS1-T TR-ACS2-T TR-ACS3-T

TR-ACS1-10F 96

TR-AC2-10F 96

TR-ACS3-10F 92

Amino Acid Level TR-ACS1-T TR-ACS2-T TR-ACS3-T

TR-ACS1-10F 93

TR-AC2-10F 82

TR-ACS3-10F 97

4.1.5. Phylogenetic Analysis of TR-ACS1-T, TR-ACS2-T, TR-ACS3-T and TR-