**4. Conclusions**

**3.4. Expression patterns of selected genes measured by sqPCR**

58 Advances in Seed Biology

Expression patterns of five genes from the seed library were studied by semi-quantitative PCR (**Figure 10**); bHLH transcription factor, three metallothioneins, antimicrobial peptide snakin and SUMO like reference gene during avocado seed development. These genes could be divided into three stages according to the time of growth of the seed in avocado fruit. The bHLH gene has an expression pattern comparable to the endogenous gene SUMO (Ubiquitin), suggesting a role throughout the formation and development of the avocado fruit seed possibly modulating the biogenesis of the seed or embryo; since from the first month of formation (E1) to ripening (E8), similar expression levels were present. For the Metallothionein gene group, PaMT3 presented a pattern of constant expression in the three stages of seed

**Figure 10.** Transcript profiling of PaMT2a, PaMT2b, PaMT3, PaSn and PabHLHS1 during seed development. (A) Total RNA was extracted from avocado seeds of 1, 4 and 8 months of development. cDNAs were synthetized and used for sqPCR , the normalizing gene was avocado ubiquitin SUMO. (B) The amount of mRNA of different genes is expressed as the ratio of the densitometric measurement of the sample RT-PCR product to the SUMO corresponding product.

**Figure 9.** Plant snakin amino acid sequences alignment. Amino acids conserved are indicated by an asterisk in the consensus sequence. The black-shaded cysteine residues are present in all sequences. The sequences included are: Avocado PaSn (KC012806); potato: StSN1 (Q948Z4), StSN2 (Q93X17); *Medicago truncatula:* MtGBRP1 (XP\_003603759), MtGBRP2 (XP\_003589486); *Capsicum annum:* CaSN1 (ACC91329); *Jatropha curcas:* JcGAST1 (ACV70139), JcLTRCOR12

(ACU30848); *Solanum lycopersicum:* SlSNP1 (ADR32106) and *Fragaria x ananassa:* FaGAST1 (AAB97006).

In this work, we identified and characterized three novel metallothioneins and one transcription factor gene from avocado nativo mexicano seeds, which are expressed abundantly during seed development. This suggests that they can have a protagonic paper during seed development and probably form a network to protect the embryo for drought stress. More studies are necessary to elucidate the paper of these genes during avocado seed-fruit development.
