3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Quantification of expression of two enzymes from the lignin
synthesis pathway on corn stalk collected at three ages
Luis Felipe P. Silva 1 and Larissa V. Março 1
1 Faculdade de Medicina Veterinária e Zootecnia, FMVZ/USP, Pirassununga, SP
1. Objetives
The objective was to identify genes responsible for lignification in maize cultivars, resulting in decrease of fiber
digestibility, since cell-wall lignification of tropical forages has been viewed as the main obstacle for microbial
fermentation in the rumen 1 .
2. Material and Methods
Fifteen hybrids from the Instituto Agronômico de Campinas breeding program were evaluated in a random block
design with three repetitions, harvested with 90, 120 and 150 days after germination. The 4 th and 5 th stalk
internodes were removed for determination of chemical composition and in vitro digestibility. Samples of the 4 th
internode above group were analyzed for in vitro NDF rumen digestibility (IVNDFD), and the cultivars were
separated in two groups with fast or slow decline of NDF digestibility, with 3 cultivars in each group.
Total RNA from 54 samples (6 cultivars, 3 blocks, 3 ages) was extracted according to the protocol of
Chomczynski e Sacchi 2 . After quantification of total RNA, the three blocks from each maize cultivar were
grouped in a single tube totaling 18 samples for DNaseI treatment and cDNA synthesis.
The quantification of gene expression was done by real-time PCR in an ABI StepOneTM Sequence Detection
System® (Applied Biosystems Foster City, CA, USA).
3. Results
There was no effect of treatment or age on the expression of the gene COMT. Expression of C4H was altered by
age, increasing C4H expression with advance in maturity (Table 1). There was a significant Treatment x Age
interaction for the expression of C4H, with expression increasing with age only at the SLOW group, composed by
three cultivars that maintained IVNDFD with advanced maturity. There was no effect of age on
the expression of C4H for the group with FAST decline of IVNDFD with advance in maturity (Tabela 1).
Table 1. Quantification of gene expression for two enzymes from the lignin synthesis pathway in maize cultivars
with either fast or slow slope of decline of rumen digestibility with advance in maturity.
Treatment Age
SLOW
FAST
Genes 1
COMT C4H
30 0.83 -3.25 A
60 0.68 -0.01 B
90 1.82 -0.18 B
30 1.53 -1.85 A
60 1.76 -0.78 A
90 0.96 0.02 A
TRT ns ns
Significance
P
AGE ns